Glucanase production and methods of using the same

ABSTRACT

Methods and compositions are described for producing a glucanase in transgenic plants and then incorporating parts of the transgenic plants in animal feed. The feed glucanase enzyme displays activity across a broad pH range, and tolerance to temperatures that are often encountered during the process of preparing animal feeds. Producing the enzyme in the transgenic plant enhances the thermal stability of the enzyme.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/599,543, which was filed on Oct. 11, 2019. U.S. patent applicationSer. No. 16/599,543 is a continuation of U.S. patent application Ser.No. 15/569,592, which was filed on Oct. 26, 2017 as 35 U.S.C. § 371national phase application of PCT/US2016/032418, which was filed May 13,2016, and claimed the benefit of U.S. Provisional Patent Application No.62/161,482 filed May 14, 2015, all of which are incorporated herein byreference as if fully set forth.

The sequence listing electronically filed with this application titled“Sequence Listing,” which was created on Jun. 23, 2021 and has a size of146,050 bytes, is incorporated by reference herein as if fully setforth.

FIELD

This disclosure relates to transgenic plants expressing glucanases withimproved thermal stability, nucleic acids encoding the same, as well asmethods of processing transgenic plants and tissues, and producing andutilizing animal feed. This disclosure also relates to feed additivesand grain and fiber processing additives that include glucanases.

BACKGROUND

The abundance of non-starch polysaccharides (NSPs) in the diets ofmonogastric and ruminant animals can adversely affect the nutritionalvalue of feed, and also present an opportunity to improve nutritionalcontent if they can be degraded in the diet or converted into beneficialnutritional components. NSPs are among the primary structural componentsof plant cell wall (cellulose, hemicellulose, xyloglucans,arabionxylans, galactans, arabinogalactans, etc.) and can also serve ascarbohydrate storage reserves in some plants. Additionally, pectins andgums are considered non-cell wall NSP. Because of their variousstructural and biological roles, NSPs often bind or encase the starch,proteins, fats and other nutrients that are present in plant-based feedingredients (such as cereals, legumes, silage etc.) and otheringredients, inhibiting the animal's ability to digest nutrientsefficiently. Increased levels of NSPs in the diet may increase viscosityof intestinal contents, which can interfere with digestive enzymes andreduce the digestibility of nutrients, thereby increasing feedconversion (mass of feed divided by the mass of meat produced) andreducing body weight gain (Iji, P. A. 1999. The impacts of cerealnon-starch polysaccharides on intestinal development and function in thebroiler chickens. Worlds Poult. Sci. J. 55:375-387, which isincorporated herein by reference as if fully set forth). For example,feeding increasing levels of guar meal germ (0, 5, or 7.5%) or guar mealhulls (0, 2.5, or 5%) to broilers resulted in increasing digestaviscosity (Lee, J. T., C. A. Bailey, and A. L. Cartwright. 2003.β-Mannanase ameliorates viscosity-associated depression of growth inbroiler chickens fed guar germ and hull fractions. Poult. Sci.82:1925-1931, which is incorporated herein by reference as if fully setforth). In addition to increasing the viscosity, body weight gain andfeed conversion was also worse with increasing guar meal hull,demonstrating the negative effects of high viscosity on animalperformance.

NSPs have also been known to inadvertently trigger immune responses inthe gut, which may further detract from efficiency of feed utilizationand have implications for animal health.

In addition to the cereal components, diets now also routinely containDDGS (dried distillers grains and solubles) that is also not easilydigested. Multiple studies have shown that enzyme supplementation canincrease diet metabolizable energy (ME), and, or, decrease the viscosityof diets containing high levels of wheat, barley, DDGSs, or otherfibrous components. The addition of carbohydrases to corn-soybeanmeal-based broiler diets, when formulated to have a 3% reduction indietary ME, has been accomplished without compromising the feedconversions of broilers reared under either hot or cool seasons. It hasbeen determined that the hydrolyzed β-d-glucan is responsible forimproved growth.

SUMMARY

In an aspect, the invention relates to a method of identifying maizeevent 4588.259, 4588.757 or 4588.652 in a sample. The method comprisescontacting a sample with a first primer and a second primer. The methodcomprises amplifying a nucleic acid in the sample to obtain an amplifiedproduct. The method also comprises detecting the amplified productspecific to a target sequence in maize event 4588.259, 4588.757 or4588.652.

In an aspect, the invention relates to an animal feedstock comprising atransgenic plant or part thereof. The transgenic plant or part thereofcomprises a synthetic nucleic acid encoding a glucanase. The glucanaseincludes an amino acid sequence with at least 70% identity to areference sequence selected from the group consisting of: SEQ ID NOS:4-6, and is capable of degrading one or more polysaccharides.

In an aspect, the invention relates to a method of producing an animalfeedstock. The method includes mixing a transgenic plant or part thereofwith plant material to form a mixture. The transgenic plant or partthereof comprises a synthetic nucleic acid encoding a glucanase. Theglucanase includes an amino acid sequence with at least 70% identity toa reference sequence selected from the group consisting of: SEQ ID NOS:4-6, and is capable of degrading one or more polysaccharides.

In an aspect, the invention relates to a method of increasingmetabolizable energy of a diet. The method includes mixing a transgenicplant or part thereof with a feed ingredient. The transgenic plant orpart thereof comprises a synthetic nucleic acid encoding a glucanasecomprising an amino acid sequence with at least 70% identity to areference sequence selected from the group consisting of: SEQ ID NOS:4-6, and is capable of degrading one or more polysaccharides.

In an aspect, the invention relates to a method of enhancing productionof fermentable sugars from grains. The method includes mixing grainsderived from any one of the transgenic plants described herein withgrains derived from a different plant to form mixed grains. The methodalso includes processing mixed grains into fermentable sugars. Thefermentable sugars are subsequently converted into ethanol or a similarfermentation product, which may include butanol, lactic acid, citricacid, acetic acid, or other fuels or chemicals.

In an aspect, the invention relates to a transgenic plant, transgenicgrain, or transgenic biomass comprising a synthetic nucleic acidencoding a glucanase. The glucanase includes an amino acid sequence withat least 70% identity to a reference sequence selected from the groupconsisting of: SEQ ID NOS: 4-6. The glucanase is capable of degradingone or more polysaccharides.

In an aspect the invention relates to a synthetic polypeptide or afragment thereof. The synthetic polypeptide or a fragment thereofcomprises an amino acid sequence with at least 70% identity to areference sequence selected from the group consisting of: SEQ ID NOS:4-6. The glucanase is capable of degrading one or more polysaccharides.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of thepresent invention will be better understood when read in conjunctionwith the appended drawings. For the purpose of illustrating theinvention, there are shown in the drawings particular embodiments. It isunderstood, however, that the invention is not limited to the precisearrangements and instrumentalities shown. In the drawings:

FIG. 1 illustrates the expression vector pAG4258 carrying a single feedglucanase expression unit.

FIG. 2 illustrates the expression vector pAG4588 carrying a single feedglucanase expression unit.

FIG. 3 illustrates the expression vector pAG4597 carrying a single feedglucanase expression unit.

FIG. 4 illustrates the expression vector pAG4708 carrying a single feedglucanase expression unit.

FIG. 5 illustrates the expression vector pAG4766 carrying two feedglucanase expression units.

FIG. 6 illustrated the expression vector pAG4767 carrying two feedglucanase expression units.

FIG. 7 illustrates the expression vector pAG4770 carrying three feedglucanase expression units.

FIG. 8 illustrates the expression vector pAG4771 carrying three feedglucanase expression units.

FIG. 9 is a chart illustrating the range of glucanase activity recoveredfrom ears of the maize plants that carried pAG4588 construct.

FIG. 10 is a chart illustrating the range of glucanase activityrecovered from ears of the maize plants that carried pAG4597 construct.

FIG. 11 is a diagram showing the T-DNA integration site in chromosome 7of maize event 4588.652.

FIG. 12 is a chart illustrating glucanase activity observed in T1plants.

FIG. 13 is a chart illustrating the glucanase activity in the seeds ofhemizygous, homozygous, and hybrid plants.

FIG. 14 illustrates general design of the real-time PCR assay used todetermine presence of the T-DNA locus (standard and real-time PCR) andzygosity (real-time PCR only) in transgenic events. Letters A, B, X andY with arrows indicate primer binding sites. Rectangular boxes A+B andX+Y represent PCR products amplified from respective primer pairs.

FIG. 15 illustrates general design of the standard PCR assay used todetermine presence of the T-DNA locus and zygosity in transgenic events.Letters A, B, and C with arrows indicate primer binding sites.Rectangular boxes A+B and A+C represent PCR products amplified fromrespective primer pairs.

FIG. 16 illustrates the standard multiplex PCR analysis of the selfedsegregating 4588.652 plants.

FIG. 17 is a graph illustrating real-time PCR data to determine presenceof the T-DNA locus and zygosity for maize event 4588.259 (FG259).

FIGS. 18A and 18B are charts illustrating glucanase activity in theGrower Diet (FIG. 18A) and the Starter Diet (FIG. 18B) before and afterpelleting.

FIGS. 19A and 19B are charts illustrating glucanase activity in wildtype (WT) flour mixed with microbial glucanase and transgenic flourproducing glucanase after heat treatment at temperatures of 130° C.(FIG. 19A) and 94° C. (FIG. 19B).

FIGS. 20 and 21 are graphs illustrating the optimum pH for measuringAGR2314 activity in an assay at 37° C. (FIG. 20) and 80° C. (FIG. 21).

FIG. 22 is a graph illustrating an example of the optimum pH of the feedglucanase that is produced in transgenic flour.

FIGS. 23A and 23B are charts illustrating glucanase activity on multiplesubstrates at 37° C. (FIG. 23A) and 80° C. (FIG. 23B).

FIGS. 24A and 24B are charts illustrating enzymatic hydrolysis ofuntreated seeds fiber of transgenic maize plants expressing AGR2314.FIG. 24A shows glucose yield and FIG. 24B shows xylose yield.

FIGS. 25A and 25B are charts illustrating enzymatic hydrolysis of seedfiber of transgenic maize plants expressing AGR2314 pretreated with thedilute acid. FIG. 25A shows glucose yield and FIG. 25B shows xyloseyield.

FIG. 26 is a chart illustrating the body weight gain (BWG) during the28-day poultry feeding trial.

FIG. 27 is a chart illustrating the changes in poultry BWG per timeinterval during 28 day feeding trial.

FIG. 28 is a chart illustrating feed consumption during the 28-daypoultry feeding trial using two different diets (corn/barley based andcorn/LF-DDGS based) with (+) or without (−) a glucanase.

FIG. 29 is a chart illustrating feed conversion rate (FCR) during the28-day poultry feeding trial with two different diets (corn/barley basedand corn/LF-DDGS based diets) with (+) or without (−) a glucanase.

FIG. 30 is a chart illustrating the effect of glucanase on poultry BWFin experimental treatment.

DETAILED DESCRIPTION OF EMBODIMENTS

Certain terminology is used in the following description for convenienceonly and is not limiting.

“Synthetic nucleic acid sequence,” “synthetic polynucleotide,”“synthetic oligonucleotide,” “synthetic DNA,” or “synthetic RNA” as usedherein refers to a nucleic acid sequence, a polynucleotide, anoligonucleotide, DNA, or RNA that differs from one found in nature byhaving a different sequence than one found in nature or a chemicalmodification not found in nature. This can include, but is not limitedto, a DNA sequence created using biotechnology tools. Such tools includebut are not limited to recombinant DNA technology, polymerase chainreaction (PCR), biotechnology mutagenesis techniques using PCR orrecombination techniques including digestion and ligation of DNA,chemical mutagenesis techniques, chemical synthesis, or directed use ofnucleases (so called “genome editing” or “gene optimizing”technologies).

“Synthetic protein,” “synthetic polypeptide,” “synthetic oligopeptide,”or “synthetic peptide” as used herein refers to a protein, polypeptide,oligopeptide or peptide that was made through a synthetic process. Thesynthetic process can include, but is not limited, to chemical synthesisor recombinant technology. The synthetic process may include productionof a protein, polypeptide, oligopeptide or peptide by expression of asynthetic nucleic acid sequence in a living cell or by in vitroexpression using a cell-free extract.

As used herein, “variant” refers to a molecule that retains a biologicalactivity that is the same or substantially similar to that of theoriginal sequence. The variant may be from the same or different speciesor be a synthetic sequence based on a natural or prior molecule.

As used herein, “alignment” refers to a plurality of nucleic acid oramino acid sequences aligned lengthwise for visual identification ofcommonly shared nucleotides or amino acids. The percentage of commonlyshared nucleotides or amino acid is related to homology or identitybetween sequences. An alignment may be determined by or used to identifyconserved domains and relatedness between the sequences. An alignmentmay be determined by computer programs such as CLUSTAL 0 (1.2.1)(Sievers et al. (2011) Molecular Systems Biology 7: 539 doi: 10.1038/msb.2011.75).

The words “a” and “one,” as used in the claims and in the correspondingportions of the specification, are defined as including one or more ofthe referenced item unless specifically stated otherwise.

In an embodiment, a synthetic nucleic acid encoding a glucanase isprovided. The synthetic nucleic acid may include a sequence with atleast 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identity to a reference sequence selected from the group consisting of:SEQ ID NO: 1 [AGR2314], SEQ ID NO: 2 [AGR2414], and SEQ ID NO: 3[AGR2514]. The encoded glucanase may be capable of degrading one or morepolysaccharides.

An embodiment includes a glucanase that includes a synthetic polypeptidehaving a sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98, 99 or 100% identity to a reference sequence selectedfrom the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5[AGR2414], and SEQ ID NO: 6 [AGR2514]. The glucanase may be capable ofdegrading one or more polysaccharides. The glucanase may be modified forimproved thermal stability.

A glucanase modified for thermal stability can be produced by standardmolecular biological techniques and then screened. The glucanase can besubjected to mutation and then screened for thermal stability. Screeningsystems that can be utilized include lambda phage, yeast, or otherexpression systems that allow production of the protein and/or testingof its physical and/or functional characteristics. From a population ofmodified proteins, candidates can be isolated and analyzed further.Further analysis may include DNA sequencing, functional assays,structural assays, enzyme activity assays, and monitoring changes inthermal stability, or structure in response to elevated temperatureconditions.

In an embodiment, a glucanase may be produced in a plant or planttissue. The glucanase may be isolated from the plant or plant tissue.

An embodiment includes a composition comprising, consisting essentiallyof, or consisting of one or more glucanases. The composition may be, butis not limited, to a transgenic plant including the one or moreglucanases, an animal feedstock or animal feed additive including theone or more glucanases or an enzyme mixture including the one or moreglucanases. A glucanase in the composition may be encoded by any one ofthe synthetic nucleic acids described herein. As used herein, the term“glucanase” refers to an enzyme capable of catalyzing the degradation ordepolymerization of complex carbohydrates.

A glucanase in the composition may be capable of degrading one or moreof disaccharides, trisaccharides, and oligosaccharides into lowermolecular weight saccharides. A glucanase in the composition may becapable of degrading one or more of cellooligosaccharide,lignocellulose, cellulose, hemicellulose, and pectin. A glucanase of thecomposition may act upon cellulose or mixed linkage beta glucans Someglucanases may have broader substrate specificities and may act on awide range of carbohydrate polymers. A glucanase of the composition mayhave enzymatic activity on a range of carbohydrate polymers. Suchenzymatic activit may be, but is not limited to, endoglucanase,exoglucanase, β-glucosidase, cellobiohydrolase, endo-1,4-β-xylanase,β-xylosidase, α-glucuronidase, α-L-arabinofuranosidase, acetylesterase,acetylxylanesterase, α-amylase, β-amylase, glucoamylase, pullulanase,β-glucanase, hemicellulase, arabinosidase, mannanase, pectin hydrolase,or pectate lyase activities. The glucanase of the composition may becapable of degrading one or more of beta-glucan, cellulose, cellobiose,pNP-D-glucopyranoside and xylan. Assays for determining activity of aglucanase for degrading various substrates are known in the art. Thebeta-glucosidase assay, endocellulase assay, exocellulase(cellobiohydrolase) assay, amylase assay, endoxylanase assay, pectinaseassay, 1,3-beta-glucosidase assay, 1,4-beta-glucosidase assay aredescribed herein in Example 13.

A glucanase of the composition may comprise, consist essentially of, orconsist of an amino acid sequence with at least 70, 72, 75, 80, 85, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a referencesequence selected from the group consisting of: SEQ ID NO: 4 [AGR2314],SEQ ID NO: 5 [AGR2414] and SEQ ID NO: 6 [AGR2514].

An embodiment includes a composition comprising, consisting essentiallyof, or consisting of an individual glucanase or a combination of two ormore glucanases herein.

In an embodiment, a glucanase of the composition may be a variant.Variants may include conservative amino acid substitutions; i.e.,substitutions with amino acids having similar properties. Conservativesubstitutions may be a polar for polar amino acid (Glycine (G, Gly),Serine (S, Ser), Threonine (T, Thr), Tyrosine (Y, Tyr), Cysteine (C,Cys), Asparagine (N, Asn) and Glutamine (Q, Gln)); a non-polar fornon-polar amino acid (Alanine (A, Ala), Valine (V, Val), Thyptophan (W,Trp), Leucine (L, Leu), Proline (P, Pro), Methionine (M, Met),Phenilalanine (F, Phe)); acidic for acidic amino acid (Aspartic acid (D,Asp), Glutamic acid (E, Glu)); basic for basic amino acid (Arginine (R,Arg), Histidine (H, His), Lysine (K, Lys)); charged for charged aminoacids (Aspartic acid (D, Asp), Glutamic acid (E, Glu), Histidine (H,His), Lysine (K, Lys) and Arginine (R, Arg)); and a hydrophobic forhydrophobic amino acid (Alanine (A, Ala), Leucine (L, Leu), Isoleucine(I, Ile), Valine (V, Val), Proline (P, Pro), Phenylalanine (F, Phe),Tryptophan (W, Trp) and Methionine (M, Met)). Conservative nucleotidesubstitutions may be made in a nucleic acid sequence by substituting acodon for an amino acid with a different codon for the same amino acid.Variants may include non-conservative substitutions. A variant may have40% glucanase activity in comparison to the unchanged glucanase. Avariant may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% activity,or an integer between any of the two values herein, in comparison to theunchanged glucanase.

In an embodiment, the one or more proteins having less than 100%identity to its corresponding amino acid sequence of SEQ ID NOS: 4-6 isa variant of the referenced protein or amino acid. In an embodiment, anisolated protein, polypeptide, oligopeptide, or peptide having asequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% identity to a protein having the sequence of any one ofSEQ ID NOS: 4-6 may be a less than full length protein having thesequence with at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% identity any one of SEQ ID NO: 4-6 along 6, 10 to 50, 10to 100, 10 to 150, 10 to 300, 10 to 400, 10 to 500, 10 to 600, 10 to700, 10 to 800, 10 to 900, or 10 to all amino acids of a protein. Thislist of sequence lengths encompasses every full length protein in SEQ IDNOS: 4-6 and every smaller length within the list, even for proteinsthat do not include over 400 amino acids. For example, the lengths of 6,10 to 50, 10 to 100, 10 to 150, 10 to 300, 10 to 400, and 10 to allamino acids would apply to a sequence with 322 amino acids. A range ofamino acid sequence lengths recited herein includes every length ofamino acid sequence within the range, endpoints inclusive. The recitedlength of amino acids may start at any single position within areference sequence where enough amino acids follow the single positionto accommodate the recited length. The range of sequence lengths can beextended by increments of 10 to 100N amino acids, where N=an integer often or greater, for sequences of 1000 amino acids or larger. Thefragment of the glucanase may be a subsequence of the polypeptidesherein that retain at least 40% activity of the glucanase. The fragmentmay have 316, 317, or 322 amino acids. The fragments may include 20, 30,40, 50, 100, 150, 200, or 300 contiguous amino acids. Embodiments alsoinclude nucleic acids encoding said amino acid sequences, and antibodiesrecognizing epitopes on said amino acid sequences.

A less than full length amino acid sequence may be selected from anyportion of one of the sequences of SEQ ID NOS: 4-6 corresponding to therecited length of amino acids. A less than full length amino acidsequence may be selected from a portion of any one of SEQ ID NOS: 4-6having a catalytic domain. The fragment may include a catalytic domainof a glucanase. For example, the catalytic domain of the glucanase ofSEQ ID NO: 4 [AGR2314] includes the following sequence:

(SEQ ID NO: 21)         10        20        30        40-GVDPFERNKILGRGINIG

ALEAPNEGDWGVVIKDEFFD         50        60        70        80IIKEAGFSHVRIPIRWSTHAAFPPYKIEPSFFKRVDEVIN        90        100      110       120 GALKRGLAVVINI

YEELMNDPEEHKERFLALWKQIADR        130        140      150       160YKDYPETLFFEIL

PHGNLTPEKWNELLEEALKVIRSID        170        180      190       200KKHTVIIGTAEWGGISALEKLRVPKWEKNAIVTIHY

NPF        210        220      230       240 EFT

QGAE W VPGSEKWLGRKWGSPDDQKHLIEEFNFIEEW       250        260      270       280 SKKNKRPIYIG

FGAYRKADLESRIKWTSFVVREAEKRGW        290        300      310 SWAY

EFCSGFGVYDPLRK

WNKDLLEALIGGDSIE

In the sequence of SEQ ID NO: 21, catalytic residues in active site areshown by enlarged characters in bold. Other active site residues thatinteract with the substrate are italicized, bold and underlined.

For example, positions 136 and 253 in SEQ ID NO: 21 are catalyticresidues in the active site, and a less than full length amino acidsequence selected from SEQ ID NO: 21 may include residues 134 and 135 atany two respective, consecutive positions within the recited length. Aless than full length amino acid sequence may be selected from a portionof any one of SEQ ID NO: 21 may have other active site residues thatinteract with the substrate. For example, positions 20, 35, 36, 135,198, 205, 210 and 286 of SEQ ID NO: 21 are the active site residues thatinteract with the substrate, and a less than full length amino acidsequence selected from SEQ ID NO: 21 may include residues 20, 35, 36,135, 198, 205, 210 and 286 at any respective, consecutive positionswithin the recited length.

A less than full length amino acid sequence may be selected from aportion of any one of SEQ ID NO: 21 may include amino acids 136-253. Aless than full length amino acid sequence may possess the glucanaseactivity. A less than full length amino acid sequence may be capable ofdegrading polysaccharides. A less than full length amino acid sequencemay contain those amino acids would contain the active site residues.

A catalytic domain may be a conserved domain. A “conserved domain”herein refers to a region in a heterologous polynucleotide orpolypeptide sequences where there is a relatively high degree ofsequence identity between the distinct sequences. With respect topolynucleotides encoding a conserved domain is preferably at least 10base pairs (bp) in length.

A conserved domain of any one of polypeptides described herein refers toa domain within a glucanase that exhibits a higher degree of sequenceidentity High degree of sequence identity may be at least 50% identity,at least 55% identity, at least 60% identity, at least 65%, at least 70%identity, at least 75% identity, at least 80% identity, at least 85%identity, at least 90% identity, at least 91% identity, at least 92%identity, at least 93% identity, at least 94% identity, at least 95%identity, at least 96% identity, at least 97% identity, at least 98%identity, at least 99% identity or at least 100% identity to consecutiveamino acid residues of a polypeptide described herein. Conserved domainsmay be identified as domains of identity to a specific consensussequence. Conserved domains may be identified by using alignmentmethods. Conserved domain may be identified with multiple sequencealignments of related proteins. These alignments reveal sequence regionscontaining the same, or similar, patterns of amino acids. Multiplesequence alignments, three-dimensional structure and three-dimensionalstructure superposition of conserved domains can be used to infersequence, structure, and functional relationships. Since the presence ofa particular conserved domain within a polypeptide is highly correlatedwith an evolutionarily conserved function, a conserved domain databasemay be used to identify the amino acids in a protein sequence that areputatively involved in functions such as degrading polysaccharides, asmapped from conserved domain annotations to the query sequence. Forexample, the presence in a protein of a sequence of SEQ ID NO: 21 thatis structurally and phylogenetically similar to one or more domains inthe polypeptides of the accompanying Sequence Listing is a strongindicator of a related function in plants. Sequences herein referred toas functionally-related and/or closely-related to the sequences ordomains of polypeptides having sequences of SEQ ID NOS: 4-6 may haveconserved domains that share at least at least ten amino acids in lengthand at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95% or 96%, 97%, 98%, or at least 99%, or about100% amino acid identity to the sequences of AGR2314, AGR2414 andAGR2514, and have similar functions that the polypeptides of the instantdescription.

In an example, sequences of AGR2314, AGR2414, and AGR2514 may be alignedas shown below.

AGR2414 sequence is a sequence of Thermotoga maritima Cel5A (3AMC inProtein Data Bank and in PubMed protein sequence database). Residuesthat interact with the substrate are underlined and are shown inenlarged bold characters: N20, H95, H96, N135, E136 (catalytic residue),Y198, H205, W210, E253 (catalytic residue), W286. (T. Wu et al. (2011),Biochim. Biophys. Acta 1814, 1.832-1840, which is incorporated herein byreference as if fully set forth). The numbering of these residues inAGR2514 is one higher because of the presence of one additional residueat the N-terminus in this sequence.

In an example, the sequences of AGR2314 and AGR2414 have 305 residuesconserved out of 317 residues and have 96% identity.

CLUSTAL O(1.2.1) multiple sequence alignment AGR2414 MGVDPFERNKILGRGINIGN ALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTHA AGR2314 MGVDPFERNKILGRGINIG NALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTHA************************************************************ AGR2414YAFPPYKIMDRFFKRVDEVINGALKRGLAVVINI HH YEELMNDPEEHKERFLALWKQIAD AGR2314QAFPPYKIEPSFFKRVDEVINGALKRGLAVVINI HH YEELMNDPEEHKERFLALWKQIAD *******   ************************************************* AGR2414RYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISALE AGR2314RYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTVIIGTAEWGGISALE********************************************:*************** AGR2414KLSVPKWEKNSIVTIHY Y NPFEFT H QGAE W VEGSEKWLGRKWGSPDDQKHLIEEFNFIEEAGR2314 KLRVPKWEKNAIVTIHY Y NPFEFT H QGAEWVPGSEKWLGRKWGSPDDQKHLIEEFNFIEE** *******:******************* ***************************** AGR2414WSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREMEKRRWSWAY W EFCSGFGVYDTLRK AGR2314WSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREAEKRGWSWAY W EFCSGFGVYDPLRK*********************************** *** *************** *** AGR2414TWNKDLLEALIGGDSIE (SEQ ID NO: 5) AGR2314QWNKDLLEALIGGDSIE (SEQ ID NO: 4)  ****************

In an example, the sequences of AGR2414 and AGR2514 below have 310residues conserved out of 318 residues and have 97% identity.

CLUSTAL O(1.2.1) multiple sequence alignment AGR2414-MGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTH AGR2514MSGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWSTH************************************:********************* AGR2414AYAFPPYKIMDRFFKRVDEVINGALKRGLAVVINIHHYEELMNDPEEHKERFLALWKQIA AGR2514AQAFPPYKIEDRFFKRVDEVINGALKRGLAVVINQ HH YEELMNDPEEHKERFLALWKQIA * ****** ************************ ************************* AGR2414DRYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISAL AGR2514DRYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISAL************************************************************ AGR2414EKLSVPKWEKNSIVTIHY Y NPFEFT H QGAE W VEGSEKWLGRKWGSPDDQKHLIEEFNFIEAGR2514 EKLRVPKWEKNAIVTIHY Y NPFEFT H QGAE WVEGSEKWLGRKWGSPDDQKHLIEEFNFIE*** *******:*********************************************** AGR2414EWSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREMEKRRWSWAY W EFCSGFGVYDTLR AGR2514EWSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREAEKRRWSWAY W EFCSGFGVYDTLR************************************ *********************** AGR2414KTWNKDLLEALIGGDSIW (SEQ ID NO: 5) AGR2514KTWNKDLLEALIGGDSIE (SEQ ID NO: 6) ******************

In an example, the sequences of AGR2314 and AGR2514 have 308 residuesconserved out of 318 residues and have 97% identity.

CLUSTAL O(1.2.1) multiple sequence alignment AGR2314-MGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEFFDIIKEAGFSHVRIPIRWSTH AGR2514MSGVDPFERNKILGRGINIG N ALEAPNEGDWGVVIKDEYFDIIKEAGFSHVRIPIRWSTH************************************:********************* AGR2314AQAFPPYKEIPSFFKRVDEVINGALKRGLAVVINI HH YEELMNDPEEHKERFLALWKQIA AGR2514AQAFPPYKIEDRFFKRVDEVINGALKRGLAVVINQ HH YEELMNDPEEHKERFLALWKQIA**********  ********************** ************************ AGR2314DRYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTVIIGTAEWGGISAL AGR2514DRYKDYPETLFFEIL NE PHGNLTPEKWNELLEEALKVIRSIDKKHTIIIGTAEWGGISAL*********************************************:************* AGR2314EKLRVPKWEKNAIVTIHY Y NPFEFT H QGAE W VPGSEKWLGRKWGSPDDQKHLIEEFNFIEAGR2514 EKLRVPKWEKNAIVTIHY Y NPFEFT H QGAE WVEGSEKWLGRKWGSPDDQKHLIEEFNFIE******************************* *************************** AGR2314EWSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREAEKRGWSWAY W EFCSGFGVYDPLR AGR2514EWSKKNKRPIYIG E FGAYRKADLESRIKWTSFVVREAEKRRWSWAY W EFCSGFGVYDTLR**************************************** *************** ** AGR2314KQWNKDLLEALIGGDSIE (SEQ ID NO: 4) AGR2514KTWNKDLLEALIGGDSIE (SEQ ID NO: 6) * ****************

Sequences that possess or encode for conserved domains that meet thesecriteria of percentage identity, and that have comparable biological andregulatory activity to the present polypeptide sequences, thus beingglucanases, described herein. Sequences having lesser degrees ofidentity, but comparable biological activity, are considered to beequivalents.

The functionality of a glucanase, variants, or fragments thereof, may bedetermined using any methods. The functionality of a glucanase may bemeasured by any one of the assays described in Example 3.

Any one or more glucanases herein may be expressed in a plant uponintroduction into the plant genome of any one more of synthetic nucleicacids described herein. The methods of introduction of synthetic nucleicacids into the plants are known in the art. The method may betransformation of the plant with a vector that includes syntheticnucleic acids.

In an embodiment, a synthetic polynucleotide having a sequence with atleast 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% identity to a reference sequence selected from the group consistingof SEQ ID NO: 7 [pAG4258], SEQ ID NO: 8 [pAG4588], SEQ ID NO: 9[pAG4597], SEQ ID NO: 10 [pAG4708], SEQ ID NO: 11 [pAG4766], SEQ ID NO:12 [pAG4767], SEQ ID NO: 13 [pAG4770], SEQ ID NO: 14 [pAG4771], SEQ IDNO: 15 [pAG4257], SEQ ID NO: 16 [pAG4692], SEQ ID NO: 17 [pAG4693], SEQID NO: 18 [pAG4705] and SEQ ID NO: 19 [pAG4706] is provided. Thesynthetic polynucleotide may include any one of the synthetic nucleicacids described herein that encode glucanase and that are capable ofdegrading a polysaccharide.

In an embodiment, a vector is provided. The vector may include any oneof the synthetic polynucleotides or nucleic acids described herein.

In an embodiment, synthetic nucleic acids are provided having a sequenceas set forth in any one of the nucleic acids listed herein or thecomplement thereof. In an embodiment, isolated nucleic acids having asequence that hybridizes to a nucleic acid having the sequence of anynucleic acid listed herein or the complement thereof are provided. In anembodiment, the hybridization conditions are low stringency conditions.In an embodiment, the hybridization conditions are moderate stringencyconditions. In an embodiment, the hybridization conditions are highstringency conditions. The hybridization may be along the length of thesynthetic nucleic acid. Examples of hybridization protocols and methodsfor optimization of hybridization protocols are described in thefollowing publications: Molecular Cloning, T. Maniatis, E. F. Fritsch,and J. Sambrook, Cold Spring Harbor Laboratory, 1982; and, CurrentProtocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston,D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl, Volume 1, John Wiley& Sons, 2000 (standard protocol) and Amersham Gene Images AlkPhos DirectLabeling and Detection System (GE Healthcare UK, Ltd), which areincorporated by reference in their entirety as if fully set forth.

In an AlkPhos Direct Labeling and Detection System, moderate conditionsmay be as follows: membranes loaded with DNA samples are prehybridizedfor at least 15 minutes at 55° C. in the hybridization buffer (12% (w/v)urea, 0.5M NaCl, 4% (w/v) blocking reagent). The labeled probe is addedto the same solution and hybridization is carried overnight at 55° C.The membranes are washed for 10 minutes at 55° C. in the primary washsolution (2M urea, 0.1% (W/v) SDS, 50 mM of 0.5M Na phosphate pH 7.0,150 mM NaCl, 1 mM of 1.0 M Mg Cl₂ and 0.2% (w/v) of blocking reagent).The washing procedure is repeated. The membranes are placed in a cleancontainer and washed for 5 minutes in a secondary buffer (1M Tris base,and 2M NaCl). The washing in the secondary solution is performed twomore time. Chemoluminescence was detected using CDP-STAR® substrate foralkaline phosphatase. Low stringency refers to hybridization conditionsat low temperatures, for example, between 37° C. and 60° C. Highstringency refers to hybridization conditions at high temperatures, forexample, over 68° C.

In the standard protocol, moderate conditions may be as follows: filtersloaded with DNA samples are pretreated for 2-4 hours at 68° C. in asolution containing 6×citrate buffered saline (SSC; Amresco, Inc.,Solon, Ohio), 0.5% sodium dodecyl sulfate (SDS; Amresco, Inc., Solon,Ohio), 5×Denhardt's solution (Amresco, Inc., Solon, Ohio), and denaturedsalmon sperm (Invitrogen Life Technologies, Inc. Carlsbad, Calif.).Hybridization is carried in the same solution with the followingmodifications: 0.01 M EDTA (Amresco, Inc., Solon, Ohio), 100 μg/mlsalmon sperm DNA, and 5-20×10⁶ cpm ³²P-labeled or fluorescently labeledprobes. Filters are incubated in hybridization mixture for 16-20 hoursand then washed for 15 minutes in a solution containing 2×SSC and 0.1%SDS. The wash solution is replaced for a second wash with a solutioncontaining 0.1×SSC and 0.5% SDS and incubated an additional 2 hours at20° C. to 29° C. below Tm (melting temperature in ° C.). Tm=81.5+16.61Log₁₀[Na⁺]/(1.0+0.7[Na⁺]))+0.41(%[G+C])−(500/n)−P-F. [Na+]=Molarconcentration of sodium ions. %[G+C]=percent of G+C bases in DNAsequence. N=length of DNA sequence in bases. P=a temperature correctionfor % mismatched base pairs (1° C. per 1% mismatch). F=correction forformamide concentration (=0.63° C. per 1% formamide). Filters areexposed for development in an imager or by autoradiography. Lowstringency conditions refers to hybridization conditions at lowtemperatures, for example, between 37° C. and 60° C., and the secondwash with higher [Na⁺] (up to 0.825M) and at a temperature 40° C. to 48°C. below Tm. High stringency refers to hybridization conditions at hightemperatures, for example, over 68° C., and the second wash with[Na+]=0.0165 to 0.0330M at a temperature 5° C. to 10° C. below Tm. In anembodiment, synthetic nucleic acids having a sequence that has at least75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identityalong its length to a contiguous portion of a nucleic acid having anyone of the sequences set forth herein or the complements thereof areprovided. The contiguous portion may be the entire length of a sequenceset forth herein or the complement thereof.

In an embodiment a synthetic nucleic acid may encode the fragment of aglucanase that have 316, 317, or 322 amino acids. The synthetic nucleicacids may encode the fragments that include 20, 30, 40, 50, 100, 150,200, or 300 contiguous amino acids and retain at least 40% activity ofthe glucanase. The functionality of a glucanase, variants, or fragmentsthereof, may be determined using any methods. The functionality of aglucanase may be measured by any one of the assays described in Example3.

Determining percent identity of two amino acid sequences or two nucleicacid sequences may include aligning and comparing the amino acidresidues or nucleotides at corresponding positions in the two sequences.If all positions in two sequences are occupied by identical amino acidresidues or nucleotides then the sequences are said to be 100%identical. Percent identity can be measured by the Smith Watermanalgorithm (Smith T F, Waterman M S 1981 “Identification of CommonMolecular Subsequences,” Journal of Molecular Biology 147: 195-197,which is incorporated by reference in its entirety as if fully setforth).

In an embodiment, synthetic nucleic acids, polynucleotides, oroligonucleotides are provided having a portion of the sequence as setforth in any one of the nucleic acids listed herein or the complementthereof. These isolated nucleic acids, polynucleotides, oroligonucleotides are not limited to but may have a length in the rangefrom 10 to full length, 10 to 800, 10 to 10 to 600, 10 to 500, 10 to400, 10 to 300, 10 to 200, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10to 60, 10 to 50, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to15, or 20 to 30 nucleotides or 10, 15, 20 or 25 nucleotides. A syntheticnucleic acid, polynucleotide, or oligonucleotide having a length withinone of the above ranges may have any specific length within the rangerecited, endpoints inclusive. In an embodiment, a hybridization probe orprimer is 85 to 100%, 90 to 100%, 91 to 100%, 92 to 100%, 93 to 100%, 94to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, 99 to 100%, or100% complementary to a nucleic acid with the same length as the probeor primer and having a sequence chosen from a length of nucleotidescorresponding to the probe or primer length within a portion of asequence as set forth in any one of the nucleic acids listed herein. Inan embodiment, a hybridization probe or primer hybridizes along itslength to a corresponding length of a nucleic acid having the sequenceas set forth in any one of the nucleic acids listed herein. In anembodiment, the hybridization conditions are low stringency. In anembodiment, the hybridization conditions are moderate stringency. In anembodiment, the hybridization conditions are high stringency.

In an embodiment, a transgenic plant comprising a synthetic nucleic acidencoding any one or more of the glucanases described herein is provided.The one or more glucanases expressed in the transgenic plant herein mayhave activity at a pH ranging from 2.0 to 10.00. The pH may be 2.0, 3.0,4.0, 5.0, 5.5, 6.0, 7.0, 7.5, 8.0, 9.0, 9.5, or 10, or a pH within arange between any two of the foregoing pH values (endpoints inclusive).The one or more glucanases expressed in a transgenic plant herein mayhave activity when exposed to a temperature in the range of 25° C. to130° C., endpoints inclusive. The temperature may be 25° C., 30° C., 35°C., 40° C., 45° C., 50° C., 55° C., 65° C., 70° C., 75° C., 80° C., 85°C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C., 120° C., 125°C., 130° C., 25° C., to 30° C., 25° C. to 35° C., 25° C. to 40° C., 25°C. to 45° C., 25° C. to 50° C., 25° C. to 55° C., 25° C. to 60° C., 25°C. to 65° C., 25° C. to 70° C., 25° C. to 75° C., 25° C. to 80° C., 25°C. to 85° C., 25° C. to 90° C., 25° C. to 95° C., 25° C. to 100° C., 25°C. to 105° C., 25° C. to 110° C., 25° C. to 115° C., 25° C. to 120° C.,25° C. to 125° C., or less than 130° C. The glucanase expressed in thetransgenic plant may have the improved activity compared to theglucanase having an identical amino acid sequence but expressed in abacterial cell. The glucanase may have improved thermal stabilitycompared to the activity of the glucanase expressed in the bacterialcell.

The one or more glucanase may be produced in any transgenic plant. Thetransgenic plant may be but is not limited to wheat, maize, soybean,barley, and sorghum.

In an embodiment, a method of making a transgenic plant that includes aglucanase is provided. The method may include contacting a plant cellwith any one of the synthetic nucleic acids herein. The syntheticnucleic acids may be part of any one of the vectors described herein.The vector may include a synthetic nucleic acid encoding a glucanase.The glucanase may comprise, consist essentially of, or consist of anamino acid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, or 100% identity to a reference sequenceselected from the group consisting of: SEQ ID NO: 4 [AGR2314], SEQ IDNO: 5 [AGR2414] and SEQ ID NO: 6 [AGR2514]. The method may also includeregenerating a transgenic plant from the transgenic plant cell. Themethod may include selecting the transgenic plant expressing aglucanase.

The transgenic plant herein is also referred to as an “event.” An eventis characterized by presence of the transgene comprising a syntheticnucleic acid encoding a glucanase. The term “event” also refers to thegenomic region of the transformed parent comprising the insertedsynthetic nucleic acid sequence and the parent genomic sequencesflanking the insertion. The term “event” also refers to progeny producedby crossing of the transgenic plant and a non-transgenic plant of thesame genetic background. The term “line” also refers to progeny producedby crossing of the transgenic plant and a non-transgenic plant with anygenetic background. After repeated crosses, the transgene and theflanking sequences of the originally transformed parent may be presentin a progeny plant in the same location in the genome or on the samechromosome as in the transformed parent.

The transgenic plant may be homozygous for the transgene comprising asynthetic nucleic acid encoding a glucanase.

The transgenic plant may be hemizygous for the transgene comprising asynthetic nucleic acid encoding a glucanase. To produce homozygousplants expressing a glucanase, a hemizygous transgenic plant may beself-crossed. Progeny may be obtained from such crosses. The progeny mayinclude homozygous, hemizygous and wild type plants. A hemizygous plantmay be phenotypically indistinguishable from the wild type plants. Themethod may include analyzing the progeny for the presence of thetransgene and selecting a progeny plant that includes the transgene. Amethod of identifying the homozygous event by PCR is described herein inExample 8.

In an embodiment, the method may further include crossing a hemizygoustransgenic plant to another transgenic plant hemizygous for the sametransgene. The method may include selecting a first progeny plant thatis homozygous for the transgene. The method may further include crossingthe transgenic plant to a wild type plant of the same, or different,genetic background. Progeny may be obtained from such crosses. Theprogeny may include hemizygous and wild type plants. The method mayinclude selecting a first progeny plant that is hemizygous for thetransgene. The method may further include selfing the first hemizygousprogeny plant and selecting a second progeny plant that is homozygousfor the transgene comprising a synthetic nucleic acid sequence encodinga glucanase.

The glucanase may have activity and improved thermal stability whenexposed to high temperatures as here described.

It has been unexpectedly discovered that expression and accumulation ofan enzyme in a plant provides the enzyme with additional thermalstability relative to the same enzyme that is produced microbially.

In an embodiment, the method of making a transgenic plant includestransformation. For transformation, the nucleic acid may be introducedinto a vector. Suitable vectors may be cloning vectors, transformationvectors, expression vectors, or virus-based vectors. The expressioncassette portion of a vector may further include a regulatory elementoperably linked to a nucleic acid encoding a glucanase. In this context,operably linked means that the regulatory element imparts its functionon the nucleic acid. For example, a regulatory element may be apromoter, and the operably linked promoter would control expression ofthe nucleic acid.

The expression of a nucleic acid encoding a glucanase from theexpression cassette may be under the control of a promoter whichprovides for transcription of the nucleic acid in a plant. The promotermay be a constitutive promoter or, tissue specific, or an induciblepromoter. A constitutive promoter may provide transcription of thenucleic acid throughout most cells and tissues of the plant and duringmany stages of development but not necessarily all stages. An induciblepromoter may initiate transcription of the nucleic acid sequence onlywhen exposed to a particular chemical or environmental stimulus. Atissue specific promoter may be capable of initiating transcription in aparticular plant tissue. Plant tissue may be, but is not limited to, astem, leaves, trichomes, anthers, cob, seed, endosperm, or embryo. Theconstitutive promoter may be, but is not limited to the maize ubiquitinpromoter (ZmUbil), Cauliflower Mosaic Virus (CAMV) 35S promoter, theCestrum Yellow Leaf Curling Virus promoter (CMP), the actin promoter, orthe Rubisco small subunit promoter. The tissue specific promoter may bethe maize globulin promoter (ZmGlb1), the rice glutelin promoter(prGTL), the maize zein promoter (ZmZ27), or the maize oleosin promoter(ZmOle). The promoter may provide transcription of a syntheticpolynucleotide having a sequence with at least 70, 72, 75, 80, 85, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to a referencesequence selected from the group consisting of SEQ ID NO: 7 [pAG4258],SEQ ID NO: 8 [pAG4588], SEQ ID NO: 9 [pAG4597], SEQ ID NO: 10 [pAG4708],SEQ ID NO: 11 [pAG4766], SEQ ID NO: 12 [pAG4767], SEQ ID NO: 13[pAG4770], SEQ ID NO: 14 [pAG4771], SEQ ID NO: 15 [pAG4257], SEQ ID NO:16 [pAG4692], SEQ ID NO: 17 [pAG4693], SEQ ID NO: 18 [pAG4705] and SEQID NO: 19 [pAG4706] and expression of glucanase that is capable ofdegrading a polysaccharide.

In an embodiment, the transformation in the method of making atransgenic plant may be stable transformation, wherein the nucleic acidencoding the glucanase integrates into the genome of the transformedplant. The transformation may be Agrobacterium-mediated transformationusing a vector suitable for stable transformation described herein. Themethod of making a transgenic plant may include any other methods fortransforming plants, for example, particle bombardment, or protoplasttransformation via direct DNA uptake. The transgenic plant may includeany synthetic nucleic acid, amino acid sequence, or vector herein.

In an embodiment, the method of making a transgenic plant may includetransient transformation to transiently express the recombinant protein.The term “transient expression” refers to the expression of an exogenousnucleic acid molecule delivered into a cell: e.g., a plant cell, and notintegrated in the plant's cell chromosome. Expression fromextra-chromosomal exogenous nucleic acid molecules can be detected aftera period of time following a DNA-delivery. Virus-based vectors may beused to carry and express exogenous nucleic acid molecules. Virus-basedvectors may replicate and spread systemically within the plant. Use ofvirus based vectors may lead to very high levels of glucanaseaccumulation in transgenic plants.

Methods of making a transgenic plant, methods of increasing utilizationof non-starch polysaccharides in an animal, methods for enhancingproduction of fermentable sugars from grains, methods for increasingmetabolizable energy of a diet, methods preparing and animal feedstockand methods for producing genetically engineered plants homozygous for asynthetic nucleic acid that encodes a glucanase may comprise a method ofdetection herein as part of making transgenic plants and/or identifyingplants, plant biomass or animal feed that comprise a synthetic nucleicacid herein.

An embodiment comprises a kit for identifying maize event 4588.259,4588.757 or 4588.652 in a sample. The kit may comprise a first primerand a second primer.

The first primer and the second primer may be capable of amplifying atarget sequence specific to an event. The target sequence may include anucleic acid with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% identity to a reference sequence selected fromSEQ ID NOS: 51-55. The target sequence may be a sequence included in ajunction between a genomic sequence of a transformed plant and asequence of the T-DNA insertion. The target sequence may be included ina sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100% identity to a reference sequence selected from SEQID NOS: 22-31.

The kit may comprise the first primer comprising a nucleic acid sequenceselected from SEQ ID NOS: a nucleic acid sequence selected from SEQ IDNOS: 38, 41, and 47. The kit may comprise the second primer comprising anucleic acid sequence selected from SEQ ID NOS: 39, 42, 43, 45, and 46.The kit may comprise the first primer comprising the nucleic acidsequence of SEQ ID NO: 38 and the second primer comprising the nucleicsequence of SEQ ID NO: 39. The kit may comprise the first primercomprising the nucleic acid sequence of SEQ ID NO: 41 and the secondprimer comprising the nucleic acid sequence of SEQ ID NO: 42. The kitmay comprise the first primer comprising the nucleic acid sequence ofSEQ ID NO: 41 and the second primer comprising the nucleic acid sequenceof SEQ ID NO: 43. The kit may comprise the first primer comprising thenucleic acid sequence of SEQ ID NO: 47 and the second primer comprisingthe nucleic acid sequence of SEQ ID NO: 45. The kit may comprise thefirst primer comprising the nucleic acid sequence of SEQ ID NO: 47 andthe second primer comprising the nucleic acid sequence of SEQ ID NO: 46.The first primer and the second primer may be capable of amplifying thetarget sequence to produce an amplified product. The amplified productor the target sequence may be capable of hybridizing to the sequence ofthe nucleic acid comprising a sequence of SEQ ID NO: 40, or SEQ ID NO:44 under conditions of high stringency. The target sequence may be usedas a probe for diagnosing any one of the events described herein.

A sample may include any sample in which nucleic acids from plant matterare present. A sample may be a protein sample. A protein sample mayinclude any sample in which proteins from plant matter are present. Thesample or protein sample may include any plant matter. The plant mattermay derive from a plant or part thereof. The plant material may derivefrom an animal feed or food.

In an embodiment, a method of identifying maize event 4588.259, 4588.757or 4588.652 in a sample is provided. The method may include contacting asample with a first primer and a second primer. The method may includeamplifying a synthetic polynucleotide comprising a target sequencespecific to the maize event. The target sequence may be any targetsequence described herein. The first primer and the second primer may becapable of amplifying the target sequence to produce an amplifiedproduct. The amplified product may be used to determine whether a plantresulted from a sexual crossing or selfing contains one or more of thetarget sequences and diagnose specific events. The length of theamplified product from the sample of the maize event may differ from thelength of the amplified product from the sample of wild type plant ofthe same genetic background. The amplified product from the event samplemay be further used as probe that hybridizes to a syntheticpolynucleotide comprising a specific region encoding a glucanase underconditions of high stringency. The method may include further detectinghybridization of the at least one probe to the specific region of thetarget sequence.

In an embodiment, an animal feedstock comprising any one or more of thetransgenic plants described herein or parts of the transgenic plants isprovided. The term “animal feedstock” refers to any food, feed, feedcomposition, diet, preparation, additive, supplement, or mixturesuitable and intended for intake by animals for their nourishment,maintenance, or growth. The glucanases included in the transgenic plantsand in the animal feedstock may be active in the gastrointestinal orrumen environment of animals. The animal may be a monogastric animal.The animal may be a ruminant animal. The monogastric animal may be butis not limited to a chicken, a turkey, a duck, a swine, a fish, a cat,or a dog. The ruminant animal may be but is not limited to cattle, acow, a steer, a sheep, or a goat. The glucanases may be active afterpreparation of the animal feed. The temperatures which feeds are exposedto during ensiling may be within range of 20° C. to 70° C. Thetemperatures which feeds are exposed to during pelleting may be withinrange of 70° C. to 130° C. The glucanases may have improved thermalstability and may retain activity after being exposed to hightemperatures during feed pelleting. The glucanase with improved thermalstability may comprise, consist essentially of, or consist of an aminoacid sequence with at least 70, 72, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% identity to a reference sequence selected fromthe group consisting of: SEQ ID NO: 4 [AGR2314], SEQ ID NO: 5 [AGR2414]and SEQ ID NO: 6 [AGR2514].

In an embodiment, a glucanase may be isolated from the transgenic plantprior to being included into the animal feedstock. The glucanase may beany one of the glucanases described herein.

In an embodiment, the animal feedstock may further include a feedsupplement. The feed supplement may be any plant material. The plantmaterial may be a non-transgenic plant or a part thereof. The plantmaterial may include an engineered plant or a mutant plant. The feedsupplement may be a mineral. The mineral may be a trace mineral. Themineral may be a macro mineral. The feed supplement may be at least onevitamin. The at least one vitamin may be a fat-soluble vitamin. The feedsupplement may include one or more exogenous enzymes. The one or moreexogenous enzymes may include a hydrolytic enzyme. The hydrolyticenzyme. The hydrolytic enzyme may be an enzyme classified under EC3.4 ashydrolase. The hydrolytic enzymes may be but are not limited toxylanases, mannanases, carbohydrases, proteases, peptidases, phytases,cellulases, lipases, phospholipases, pectinases, galactosidases,laccases, amylases, hemicellulases, or cellobiohydrolases. Thehydrolytic enzymes may be expressed in the engineered plants or partsthereof included in the feed supplement. The feed supplement may includepurified hydrolytic enzymes. The feed supplements may be but are notlimited to growth improving additives, coloring agents, flavorings,stabilizers, limestone, stearine, starch, saccharides, fatty acids, or agum. The coloring agents may be carotenoids. The carotenoids may be butare not limited to cantaxanthin, beta-carotene, astaxanthin, or lutein.The fatty acids may be polyunsaturated fatty acids. The polyunsaturatedfatty acids may include but are not limited to arachidonic acid,docosohexaenoic acid (DHA), eicosapentaenoic acid (EPA) orgamma-linoleic acid. The feed supplement may be a non-transgenic plantor a part thereof. The non-transgenic plant or part thereof may includeat least one component selected from the group consisting of: barley,wheat, rye, oat, corn, rice, triticale beet, sugar beet, spinach,cabbage, quinoa, corn meal, corn pellets, corn oil, distillers grains,forage, wheat meal, wheat pellets, wheat grain, barley grain, barleypellets, soybean meal, soybean oilcake, lupin meal, rapeseed meal,sorghum grain, sorghum pellets, rapeseed, sunflower seed, and cottonseed.

The feed supplement may include at least one component selected from thegroup consisting of: soluble solids, fat, vermiculite, limestone, plainsalt, DL-methionine, L-lysine, L-threonine, monensin sodium COBAN®PREMIX, vitamin premix, inorganic feed phosphates, monocalciumphosphate, dicalcium phosphate, tricalcium phosphate, monodicalciumphosphate, rock phosphate, selenium premix, choline chloride, sodiumchloride, and mineral premix.

The feed supplement may include fish meal, fish oil, bone meal, feathermeal and animal fat. The feed supplement may include yeast or yeastextract.

In an embodiment, a method of producing an animal feedstock is provided.The method may comprise including a transgenic plant that includes anyone or more glucanase described herein in the animal feedstock. Theanimal feedstock may comprise, consist essentially of or consist of thetransgenic plant. The method may further include combining thetransgenic plant with a feed supplement. The feed supplement may be anon-transgenic plant or a part thereof. The transgenic plant may beproduced by any one of the methods described herein. The feed supplementmay be a mineral. The supplement may include one or more exogenousenzymes. The exogenous enzymes may be but are not limited to xylanases,mannanases, carbohydrases, proteases, peptidases, phytases, cellulases,lipases, phospholipases, pectinases, galactosidases, laccases, amylases,hemicellulases, or cellobiohydrolases.

In an embodiment, a method of meat production is provided. The methodmay include feeding an animal feedstock or one produced by any of themethods described herein to the animal. The method may include preparingan animal feedstock that includes a transgenic plant expressing aglucanase.

In an embodiment, a method of feeding an animal is provided. The methodmay include feeding an animal feedstock or one produced by any of themethods described herein to the animal. The method may include preparingan animal feedstock that includes a transgenic plant expressing aglucanase.

In an embodiment, a method of increasing utilization of non-starchpolysaccharides in an animal is provided. The method may include feedingan animal with an animal feedstock that includes any one or more of thetransgenic plants described herein. The method may include preparing theanimal feedstock.

In an embodiment, a method of decreasing gastrointestinal viscosity inan animal is provided. The method may include feeding an animal with ananimal feedstock that includes any one or more of the transgenic plantsdescribed herein. The method may include preparing the animal feedstock.

Addition of exogenous enzymes collectively known as carbohydrases mayameliorate the effects of non-starch polysaccharides (NSPs) in the dietof an animal. An animal feedstock that includes any one or more ofglucanases described herein may increase utilization of NSPs by theanimal that ingested the feedstock, or may decrease the anti-nutritionaleffects of the NSP on the animal that ingested the feedstock, andimprove growth of the animal. Preparing the animal feedstock may includecombining one or more transgenic plant herein with any other feedsupplement. The glucanase may be isolated, purified and added to theanimal feedstock as a pure glucanase. The glucanase may be added to theanimal feedstock in admixture with other feed supplements. Thetransgenic plant including the glucanase or the purified glucanase maybe combined with other feed supplements to form premixes.

An animal feedstock may be produced as mash feed. The animal feedstockmay be produced as pellets. The milled feed stuffs may be mixed with thepremix that includes any one of the transgenic plants that include aglucanase. The milled feed stuffs may include the plant material and thefeed supplements described herein. The feed supplements may include oneor more exogenous enzymes described herein. Enzymes may be added asliquid or solid formulations. For mash feed, a solid or liquid enzymeformulation may be added before or during the mixing step. For pelletedfeed, the enzyme preparation may be added before or after the pelletingstep. The glucanase may be included in a premix. The premix may alsoinclude vitamins and trace minerals. Macro minerals may be addedseparately to animal feedstock.

In an embodiment, a method of increasing metabolizable energy of a dietis provided. Metabolizable energy (ME) refers to the net energy of adiet or feed that is available to an animal after the utilization ofsome energy in the processes of digestion and absorption and the loss ofsome of the material as being undigested or indigestible. Metabolizableenergy may be apparent metabolizable energy (AME) measured as thedifference between the calories of the feed consumed by an animal andexcrements collected after feed consumption. Metabolizable energy may betrue metabolizable energy (TME), which is similar to AME except that italso takes into account endogenous energy. Energy contents in a diet orfeed ingredients may be determined using one of several methodologies(NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad.Press, Washington, D.C., which is incorporated herein by reference as iffully set forth). Gross energy (GE) is direct measurement using anadiabatic bomb calorimeter, which measures the heat of combustion of asample within a high oxygen atmosphere. Apparent digestible energy (DE)is GE of a feed or feedstuff minus GE of feces only. Apparentmetabolizable energy (AME) is GE of a feed or feedstuff minus GE offeces, urine, and gaseous products from digestion. For poultry, thegaseous release is very low, and typically neglected due to its verysmall value, and the urine and feces are excreted together and are notcollected separately in most cases. True metabolizable energy (TME)accounts for only the GE from excreta that is from the feed or feedstufforigin, by subtracting the endogenous energy loss from non-feed origin(i.e. sloughing of intestinal tract cells). Another energy measurementused for feedstuffs in animals is net energy (NE) which adjusts for heatincrement. Since heat increment is dependent on level of productivity,which fluctuates in poultry because of short lifespan, this variable isnot frequently used in poultry.

The TME rooster assay may be used to account for endogenous (non-feed)losses of GE by including a fasted rooster and collecting excreta tocorrect the GE from the fed (feed/feedstuff) rooster. See Sibbald, 1976,Poultry Science 55: 303-308, which is incorporated herein by referenceas if fully set forth. This assay has commonly been used for determiningTME of individual feedstuffs rather than complete feed, and requirescecetomized roosters (ceca surgically removed) to always be on hand. Theassay involves force-feeding (into the crop) a known quantity of aningredient (in birds that were previously fasted 24-48 hr) and thencollect feces for a 24-48 hour period. The equation used to calculateTME is given as TME={(GE_(f)×FI)−[(GE_(e)×EO)⁺−(GE_(e)×EO)⁻]}/FI, whereGross Energy (GE) is determined by bomb calorimetry in kcal/kg; FI isfeed intake (kg); EO is excreta output fed birds (kg); GE_(e) is theGross Energy of the excreta content; GE_(f) is the Gross Energy of thefeed; “k” signifies the quantity is from the fed birds energy output;and “−” signifies that the quantity is from the fasted birds energyoutput. The roosters (or turkeys) used in TME assays are adult birdswith a fully developed digestive tract. Research has shown that thereare differences in ME determinations using roosters (layer breeds),turkeys and broilers when analyzing same feed ingredients (Cozannet etal, 2010 J. Anim, Sci., 88(7):2382-2392, which is incorporated herein byreference as if fully set forth). So determining TME or AME usingrooster model may not be equivalent to what is observed in a youngbroiler, but is a commonly used proxy in research and industry.

For broilers, the AME assay may be used for determining complete feedand some energy supplying feedstuffs, as well as the effect from addingfeed ingredients that aid in digestion. There are two common methods fordetermining ME: 1) doing a total excreta collection and weighing andrecord feed consumption during the time period (Equation 1 below) or 2)using an indigestible marker in feed (chromic oxide, titanium oxide oracid insoluble ash) and taking a subsample of feces with no weighingrequired (Equation 2 below). The marker method of AME determination maybe used, in which no weighing of feed consumption or total fecalcollection and no need to separate feed spilled from feces pan arerequired. With the marker method, birds are fed the marker for at leasttwo days (but preferably five or more days). Feces are collected overseveral days (e.g., three days) with daily collection composited intoone sample.

AME using the total collection method (Equation 1) is calculated asfollows:

AME=[(GE _(f) ×FI)−(GE _(e) ×EO)]/FI,

where Gross Energy (GE) is measured in bomb calorimetry (kcal/kg); FI isfeed intake (kg); EO is excreta output (kg); e refers to excretacontent; and f refers to the feed content. AME using the marker methodis calculated as AME=[(GE_(e)/M_(e))−(GE_(f)/M_(f))]/(GE_(e)/M_(e)),where Gross Energy is GE (kcal/kg); M is the marker (ppm or %);“e”=excreta content; “f”=feed content.

Another method that may be used to determine AME of feed wheninvestigating feed additives that aid in digestion is Ileal digestibleenergy (IDE). This method uses the AME marker method (described above),but the birds are euthanized and a section of ileum excised and contentsremoved, dried and analyzed for GE and the marker. The IDE method may beused effectively for testing and comparing feed additives used toimprove digestion/absorption of feed energy. The benefit of IDE, is nocages with collection pans are required and can collect during afloor-pen study. With the marker method, birds are fed the marker for atleast two days (and preferably five or more days).

AME using the IDE marker method (Equation 2) is calculated as follows:

AME=GE _(f)−(GE _(d) ×M _(f) /M _(d)),

where GE (kcal/kg); M represents the marker; “_(d)” represents thedigesta content; and “_(f)” signifies the feed content.

AME and TME may be corrected for nitrogen retention (AMEn and TMEn). Toadjust, the grams of N are multiplied by 8.22 kcal/g (GE of uric acid;primary excretory product of protein tissue oxidized for energy), whichalso is subtracted off of the GE consumed. See Hill, F. W., and D. L.Anderson, 1958, “Comparison of metabolizable energy and productiveenergy determinations with growing chicks.” J. Nutr. 64:587-603, whichis incorporated herein by reference as if fully set forth. Calculationsfor total collection of marker method for AMEn are shown in Equation 3and Equation 4 below, respectively. Equation 3: AMEn, total collection:

AMEn={(GE _(f) ×FI)−(GE _(e) ×EO)−[8.22×(N _(f) −N _(e))]}/FI,

where GE=kcal/kg; FI=feed intake (kg); EO=excreta output (kg);N=nitrogen (g); e=excreta content; f=feed content.

Equation 4: IDEn, marker method:

AMEn=GE _(f)−(GE _(d) ×M _(f) /M _(d))−{8.22×[N _(f)−(Nd×M _(f) /M_(d))]},

where GE=kcal/kg; M=marker; N=nitrogen (g/kg) “d”=digesta content;“f”=feed content.

While the TME method may be used for determining ME of individualingredients, the AME (IDE) method may be used with broilers to measureME in individual ingredients or total diet and testing effects improvingME by use of enzymes or other feed additives.

A diet or feed may include any feed ingredient or mixture of ingredientsincluding water. The diet may be any food, feed, feed composition, diet,preparation, additive, supplement, or mixture included in an animalfeedstock described herein. The diets are known in the art and describedat least in the following publications: Nutrient Requirements ofPoultry, 1994, National Research Council, National Academy Press,Washington, D.C.; Broiler Performance and Nutrition Supplement,Cobb-500™, L-2114-07EN, July, 2015; Broiler Performance and NutritionSupplement, Cobb-700™, L-21124-13EN, Dec. 21, 2012; Broiler Performanceand Nutrition Supplement, CobbAvian™ L-2144-04EN, April, 2012; BroilerPerformance and Nutrition Supplement, CobbSasso™, L-2154-01, May 7,2008; Ross 308 Broiler: Nutrition Specifications, 2014 Aviagen,0814-AVNR-035; Ross Nutrition Supplement 2009, Aviagen; Ross 708Broiler: Nutrition Specification, 2014 Aviagen, 0814-AVNR-036; Ross PM3Brioler Nutrition Specification, 2014 Aviagen, 0814-AVNR-037; ArborAcres Plus Broiler Nutrition Specifications, 2014 Aviagen,1014-AVNAA-043; Arbor Acres Broiler Nutrition Supplement, 2009 Aviagen;and Association of American Feed Control Officials (AAFCO) 2015 OfficialPublication, Nutrient Requirements for Poultry, all of which areincorporated herein by reference as if fully set forth.

In an embodiment, the diet may be a diet for broilers. The diet forbroilers may be composed of one or more of the following ingredients:51.49% (w/w)-61.86% (w/w) corn, 25.45% (w/w)-35.03% (w/w) soybean meal,5.00% (w/w) corn distillers dry grains plus soluble solids, 2.00% (w/w)vermiculite, 0.30% (w/w)-1.99% (w/w) dicalcium phosphate, 1.00% (w/w)poultry fat, 0.81% (w/w)-4.01% (w/w) limestone, 0.24% (w/w)-0.50% (w/w)salt (NaCl), 0.13% (w/w)-0.45% (w/w) DL-methionine, 0.20% (w/w) cholinechloride 60, 0.20% (w/w) mineral premix, 0.05% (w/w) vitamin premix,0.13% (w/w)-0.23% (w/w) L-lysine, 0.08% (w/w)-0.14% (w/w) L-threonine,0.05% (w/w) coban, 0.05% (w/w) selenium premix, 0.15% (w/w) sodiumbicarbonate and 0.10% (w/w) sand. Digestible lysine in the diet may be1.00% (w/w) to 1.20% (w/w). Digestible methionine in the diet may be0.47% (w/w) to 0.54% (w/w). Digestible methionine and cysteine in thediet may be 0.98% (w/w) to 1.10% (w/w). Digestible threonine in the dietmay be 0.68% (w/w) to 0.84% (w/w). Digestible tryptophan in the diet maybe 0.17% (w/w) to 0.22% (w/w). Calcium in the diet may be 0.71% (w/w) to0.96% (w/w). Available phosphorus in the diet may be 0.17% (w/w) to0.46% (w/w). Sodium in the diet may be 0.17% (w/w) to 0.19% (w/w). Theconcentration of each ingredient within any one of the ranges herein maybe any value between any two of the concentration points included in therange. In an embodiment, the diet may be the diet for broilers composedof one or more of the following ingredients: 30.00% (w/w)-75.00% (w/w)corn, 5.00% (w/w)-75.00% (w/w) wheat; 5.00% (w/w)-65.00% (w/w) barley;5.00% (w/w)-30.00% (w/w) sorghum, 5.00% (w/w)-50.00% (w/w) millet,10.00% (w/w)-45.00% (w/w) soybean meal, 5.00% (w/w)-20.00% (w/w) Canola(Rapeseed) meal, 2.00% (w/w)-15.00% (w/w) corn gluten meal, 5.00%(w/w)-15.00% (w/w) sunflower meal, 5.00% (w/w)-30.00% (w/w) corndistillers dry grains plus soluble solids, 1.00% (w/w)-8.00% (w/w)poultry/porcine/bovine meat and bone meal, 1.00% (w/w)-8.00% (w/w) fishmeal, 0.10% (w/w)-2.1% (w/w) dicalcium or monocalcium or defluorinatedphosphate, 0.50% (w/w)-6.00% (w/w) soy oil or vegetable oil or animalfat or grease or combination, 0.81% (w/w)-2.00% (w/w) limestone, 0.50%(w/w)-7.00% soy hulls, 0.24% (w/w)-0.50% (w/w) salt (NaCl), 0.13%(w/w)-0.50% (w/w) DL-methionine, 0.01% (w/w)-0.20% (w/w) cholinechloride 60, 0.10% (w/w)-0.20% (w/w) mineral premix, 0.05% (w/w)-0.25%(w/w) vitamin premix, 0.05% (w/w)-0.30% (w/w) L-lysine, 0.10%(w/w)-0.30% (w/w) DL-Methionine or methionine analog (MHA), 0.05%(w/w)-0.20% (w/w) L-threonine, 0.05% (w/w) coban, 0.05% (w/w) seleniumpremix, 0.05% (w/w)-0.15% (w/w) sodium bicarbonate and 250 FTU/kg-2000FTU/kg Phytase. Metabolizable energy of the diet may be 1225(kcal/lb)-1491 (kcal/lb). Crude protein (CP) in the diet may be 15%(w/w) to 25% (w/w). Digestible lysine in the diet may be 0.85% (w/w) to1.30% (w/w). Digestible methionine in the diet may be 0.45% (w/w) to0.70% (w/w). Digestible methionine and cystine in the diet may be 0.65%(w/w) to 1.10% (w/w). Digestible threonine in the diet may be 0.60%(w/w) to 0.84% (w/w). Digestible tryptophan in the diet may be 0.10%(w/w) to 0.25% (w/w). Calcium in the diet may be 0.68% (w/w) to 1.10%(w/w). Available phosphorus in the diet may be 0.17% (w/w) to 0.50%(w/w). Sodium in the diet may be 0.17% (w/w) to 0.19% (w/w). Phytase inthe diet may be 500 FTU/kg (w/w) to 8,000 FTU/kg (w/w). Theconcentration of each ingredient within any one of the ranges herein maybe any value between any two of the concentration points included in therange. Variations in the concentrations of these ingredients may also beused in a diet.

The method may include mixing a transgenic plant or part thereof with afeed ingredient to obtain a mixture. The feed ingredient may be one ormore ingredients included in the diet described herein. The transgenicplant or part thereof may be any transgenic plant or part thereofdescribed herein. The method may include feeding an animal with themixture. The body weight gain (BWG) in an animal fed with the mixturecomprising a glucanase may be higher than the BWG in a control animalfed with identical feed ingredients not mixed with a transgenic plantincluding a glucanase. In an embodiment, the BWG in an animal fed withthe mixture comprising a glucanase may be similar to the BWG in acontrol animal fed with a high energy diet or a diet that includes moreor higher concentrations of the ingredients compared to the mixtureincluding a glucanase. In an embodiment, the feed conversion ratio (FCR)in an animal fed with the mixture comprising a glucanase may be lowerthan the FCR in a control animal fed with identical feed ingredients notmixed with a transgenic plant including a glucanase. The FCR is definedas the mass of the feed eaten by the animal divided by the animal'smass. In an embodiment, the FCR in an animal fed with the mixturecomprising a glucanase may be similar to the FCR in a control animal fedwith a high energy diet or a diet that includes more or higherconcentrations of the ingredients compared to the mixture including aglucanase.

In an embodiment, a method of enhancing thermal stability of a glucanaseis provided. The method may include producing a transgenic plant thatincludes a synthetic nucleic acid encoding the glucanase. The syntheticnucleic acid may include any one the sequences described herein. Theglucanase may be thermally stable upon exposure to temperatures in therange of 70° C. to 130° C., endpoints inclusive.

The glucanase may be thermally stable upon exposure to temperatures inthe range of 25° C. to 130° C., endpoints inclusive. The glucanase maybe thermally stable upon exposure to temperatures in the range from 25°C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 65° C., 70° C., 75°C., 80° C., 85° C., 90° C., 95° C., 100° C., 105° C., 110° C., 115° C.,120° C., 125° C., 130° C., 25° C., to 30° C., 25° C. to 35° C., 25° C.to 40° C., 25° C. to 45° C., 25° C. to 50° C., 25° C. to 55° C., 25° C.to 60° C., 25° C. to 65° C., 25° C. to 70° C., 25° C. to 75° C., 25° C.to 80° C., 25° C. to 85° C., 25° C. to 90° C., 25° C. to 95° C., 25° C.to 100° C., 25° C. to 105° C., 25° C. to 110° C., 25° C. to 115° C., 25°C. to 120° C., 25° C. to 125° C., or less than 130° C. The glucanase maybe thermally stable upon exposure to temperatures in the range of 70° C.to 130° C., endpoints inclusive. The glucanase may be thermally stableupon exposure to temperatures in the range from 70° C. to 75° C., 70° C.to 80° C., 70° C. to 85° C., 70° C. to 90° C., 70° C. to 95° C., 70° C.to 100° C., 70° C. to 105° C., 70° C. to 110° C., 70° C. to 115° C., 70°C. to 120° C., or 70° C. to 130° C., endpoints inclusive.

The above mentioned synthetic nucleic acids may be provided inembodiments herein alone, as part of another nucleic acid, as part of avector or as stated above as part of a transgenic plant.

In an embodiment, the transgenic plant may be derived from one of corn,rye, switchgrass, miscanthus, sugarcane or sorghum. The transgenic plantmay be made by Agrobacterium mediated transformation using a vectorhaving a nucleic sequence as set forth above.

In an embodiment, a method for enhancing production of fermentablesugars from grains is provided. The method may include mixing grainsderived from any one of the transgenic plants described herein withgrains from a different plant to form mixed grains. The different plantmay be a non-transgenic plant. The different plant may be an engineeredplant that includes a synthetic nucleic acid encoding at least onehydrolytic enzyme. The hydrolytic enzyme may be but is not limited toxylanase, an amylase, an endoglucanase, an exoglucanase, a feruloylesterase, a glucoamylase, an intein-modified amylase, an intein-modifiedxylanase, an intein-modified endoglucanase, an intein-modifiedexoglucanase, an intein-modified feruloyl esterase, a protease, anintein-modified protease, a phytase, or an intein-modified phytase. Themethod may include processing the mixed grains. The processing mayinclude one or more operations selected from the group consisting ofharvesting, baling, grinding, milling, chopping, size reduction,crushing, pellitizing, extracting a component from the mixed grains,purifying a component or portion of the mixed grains, extracting orpurifying starch, hydrolyzing polysaccharides into oligosaccharides ormonosaccharides, ensiling, mixing with silage or other biomass andensiling, fermentation, chemical conversion, and chemical catalysis. Thebiomass may be but is not limited to hay, straw, stover, silage,compressed and pelleted feeds, soybeans, sprouted grains, legumes, feedgrains, maize, rice, barley or wheat grains. The biomass may be anybiomass derived from agricultural waste. The method may includeconverting fermentable sugars into a biochemical product. Thebiochemical product may be but is not limited to ethanol, butanol,lactic acid, citric acid, and acetic acid.

In an embodiment, a method for reducing the viscosity of a grain mixtureis provided. The method may include mixing grains derived from any oneof the transgenic plants described herein with grains from a differentplant to form mixed grains. Water may be added to the mixed grains toform the grain mixture. The viscosity of the grain mixture may be lowerwhen it includes any one of the glucanases described herein. Theviscosity may be intestinal viscosity, which is typically measured froman intestinal sample removed from a bird or pig after euthanization. Inthis method, the digesta sample is centrifuged and the viscosity ofsupernatant is analyzed using a viscometer. For example, as describe byLee et al., ileal digesta was centrifuged for 10 min at 3,500× gravityand 0.5 ml of supernatant was put in a Brookfield Cone and PlateViscometer¹ with a CPE-40 spindle. See Lee, J. T., C. A. Bailey, and A.L. Cartwright. 2003. β-Mannanase ameliorates viscosity-associateddepression of growth in broiler chickens fed guar germ and hullfractions. Poult. Sci. 82:1925-1931, which is incorporated herein byreference as if fully set forth. Samples are analyzed for 30 sec at 40°C. and 5 rpm, to determine centipoise (cP units) readings. The higherthe cP, the higher the viscosity of the sample.

The different plant may be a non-transgenic plant. The different plantmay be an engineered plant that includes a synthetic nucleic acidencoding at least one hydrolytic enzyme. The hydrolytic enzyme may bebut is not limited to xylanase, an amylase, an endoglucanase, anexoglucanase, a feruloyl esterase, a glucoamylase, an intein-modifiedamylase, an intein-modified xylanase, an intein-modified endoglucanase,an intein-modified exoglucanase, an intein-modified feruloyl esterase, aprotease, an intein-modified protease, a phytase, or an intein-modifiedphytase. The method may include processing the grain mixture. Theprocessing may include one or more operations selected from the groupconsisting of harvesting, grinding, milling, size reduction, crushing,heating, gelotinzing, liquefaction, extracting a component from themixed grains, purifying a component or portion of the mixed grains,extracting or purifying starch, hydrolyzing polysaccharides intooligosaccharides or monosaccharides, saccharifying, fermentation,chemical conversion, and chemical catalysis.

In an embodiment, a method for enhancing ethanol production from grainsis provided. The method includes performing any one of the methods forenhancing production of fermentable sugars described herein.

The following list includes particular embodiments of the presentinvention. But the list is not limiting and does not exclude alternateembodiments, as would be appreciated by one of ordinary skill in theart.

EMBODIMENTS

1. A transgenic plant comprising a synthetic nucleic acid encoding aglucanase, wherein the glucanase includes an amino acid sequence with atleast 70% identity to a reference sequence selected from the groupconsisting of: SEQ ID NOS: 4-6, and is capable of degrading one or morepolysaccharides.2. The transgenic plant of embodiment 1, wherein the one or morepolysaccharides is selected from the group consisting of beta-glucan,cellulose, cellobiose, pNP-D-glucopyranoside and xylan.3. The transgenic plant of any one or both of the preceding embodiments,wherein the glucanase is active upon expression in the plant andexposure to a pH in the range from 4.0 to 10.0.4. The transgenic plant of any one or more of the preceding embodiments,wherein the glucanase is active upon expression in the plant andexposure to a temperature in the range from 25° C. to 130° C.5. The transgenic plant of any one or more of the preceding embodiments,wherein the glucanase activity has improved stability upon expression inthe plant compared to the activity of a glucanase having an identicalamino acid sequence and expressed in a bacterial cell.6. The transgenic plant of any one or more of the preceding embodiments,wherein a plant is selected from the group consisting of: wheat, maize,barley, rice, and sorghum.7. A transgenic plant comprising a synthetic nucleic acid including asequence with at least 70% identity to a reference sequence selectedfrom the group consisting of: SEQ ID NOS: 1-3, wherein the glucanase iscapable of degrading one or more polysaccharides.8. The transgenic plant of embodiment 7, wherein the one or morepolysaccharides is selected from the group consisting of beta-glucan,cellulose, cellobiose, pNP-D-glucopyranoside and xylan.9. The transgenic plant of any one or more of embodiments 7-8, whereinthe glucanase is active upon expression in the plant and exposure to apH in the range from 4.0 to 10.0.10. The transgenic plant of any one or more of embodiments 7-9, whereinthe glucanase is active upon expression in the plant and exposure to atemperature in the range from 25° C. to 130° C.11. The transgenic plant of any one or more of embodiments 7-10, whereinthe glucanase activity has improved stability upon expression in theplant compared to the activity of a glucanase having an identical aminoacid sequence and expressed in a bacterial cell.12. The transgenic plant of any one or more of embodiments 7-11, whereinthe transgenic plant is a plant is selected from the group consistingof: wheat, maize, barley, rice, and sorghum.13. The transgenic plant of any one or more of embodiments 7-12, whichcomprises the nucleic acid sequence of SEQ ID NO: 1 and produces anamplicon for diagnosing event 4588.259, 4588.757, or 4588.652.14. A synthetic nucleic acid comprising a sequence with at least 70%identity to a reference sequence selected from the group consisting of:SEQ ID NOS: 1-3, wherein the glucanase is capable of degrading one ormore polysaccharides.15. The synthetic nucleic acid of embodiment 14, wherein the one or morepolysaccharides is selected from the group consisting of beta-glucan,cellulose, cellobiose, pNP-D-glucopyranoside and xylan.16. A synthetic polynucleotide comprising a sequence with at least 70%identity to a reference sequence selected from the group consisting ofSEQ ID NO: 7-19, wherein the synthetic polynucleotide comprises asynthetic nucleic acid encoding a glucanase that is capable of degradingone or more polysaccharides.17. The synthetic polynucleotide of embodiment 16, wherein the one ormore polysaccharides is selected from the group consisting ofbeta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.18. A vector comprising a synthetic polynucleotide, or a fragment of asynthetic polynucleotide, of embodiment 17.19. A method of making a transgenic plant that includes a glucanasecomprising:

contacting a plant cell with a synthetic nucleic acid encoding an aminoacid sequence with at least 70% identity to a reference sequenceselected from the group consisting of: SEQ ID NOS: 1-3, wherein theglucanase is capable of degrading one or more polysaccharides;

-   -   regenerating a transgenic plant from the transgenic plant cell;        and

selecting the transgenic plant expressing a glucanase, wherein theglucanase is active and thermally stable upon exposure to a temperaturein the range from 25° C. to 130° C.

20. The method of embodiment 19, wherein the one or more polysaccharideis selected from the group consisting of beta-glucan, cellulose,cellobiose, pNP-D-glucopyranoside and xylan.21. The method of any one or both of embodiments 19-20, wherein thesynthetic nucleic acid is part of a vector of embodiment 13.22. An animal feedstock comprising a transgenic plant or part thereof ofany one or more of embodiments 1-13, the product of any one or more ofembodiments, 19-21, or a synthetic polypeptide of any one or more ofembodiments 51-54.23. The animal feedstock of embodiment 22 further comprising a feedsupplement or feed additive.24. The animal feedstock of any one or both of embodiments 22-23,wherein the feed supplement is plant material.25. The animal feedstock of any one or more of embodiments 22-24,wherein the plant material is a non-transgenic plant.26. The animal feedstock of any one or more of embodiments 22-24 whereinthe plant material is an engineered plant.27. The animal feedstock of any one or more of embodiments 22-26,wherein the feed supplement includes one or more exogenous enzymes.28. The animal feedstock of embodiment 27, wherein the one or moreexogenous enzyme includes a hydrolytic enzyme selected from the groupconsisting of: xylanase, endoglucanase, cellulase, exoglucanase,feruloyl esterase, an intein-modified xylanase, an intein-modifiedendoglucanase, an intein-modified cellulase, an intein-modifiedexoglucanase, an intein-modified feruloyl esterase, mannanase, amylase,an intein-modified amylase, phytase, an intein-modified phytase,protease, and an intein-modified protease.29. The animal feedstock of any one or more embodiments 22-28, whereinthe plant material includes at least one component selected from thegroup consisting of: forage, biomass, corn meal, corn pellets, wheatmeal, wheat pellets, wheat grain, barley grain, barley pellets, soybeanmeal, soybean oilcake, silage, sorghum grain and sorghum pellets.30. The animal feedstock of any one or more of embodiments 23-29,wherein the feed supplement includes at least one component selectedfrom the group consisting of: soluble solids, fat and vermiculite,limestone, plain salt, DL-methionine, L-lysine, L-threonine, COBAN®,vitamin premix, dicalcium phosphate, selenium premix, choline chloride,sodium chloride, and mineral premix.31. A method of producing an animal feedstock comprising mixing 1) atransgenic plant or part thereof of any one or more of embodiments 1-13,2) the product of any one or more of embodiments 19-41, or 3) asynthetic polypeptide of any one or more of embodiments 51-54 with plantmaterial.32. The method of embodiment 31 further comprising pelletizing themixture.33. The method of embodiment 32 further comprising adding a feedsupplement to the mixture.34. The method of embodiment 33, wherein the feed supplement includes atleast one exogenous enzyme.35. The method of embodiment 34, wherein the at least one exogenousenzyme includes a hydrolytic enzyme selected from the group consistingof xylanase, endoglucanase, cellulase, exoglucanase, feruloyl esterase,an intein-modified xylanase, an intein-modified endoglucanase, anintein-modified exoglucanase, an intein-modified cellulase, anintein-modified feruloyl esterase, amylase, an intein-modified amylase,mannanase, phytase, and protease.36. A method of increasing utilization of non-starch polysaccharides inan animal comprising feeding an animal with an animal feedstock 1)including a transgenic plant of any one or more of embodiments 1-13, 2)of any or more of embodiments 22-30, 3) produced by the method of anyone or more of embodiments 31-35, or 4) including a syntheticpolypeptide of any one or more of embodiments 51-54.37. The method of embodiment 36 further comprising preparing the animalfeedstock.38. The method of any or both of embodiments 36-37, wherein the animalis a monogastric animal or a ruminant animal.39. A method of enhancing thermal stability of a glucanase comprisingproducing a transgenic plant that includes a synthetic nucleic acidcomprising, consisting essentially of, or consisting of an amino acid asequence having 70% identity to a reference sequence of selected fromthegroup consisting of: SEQ ID NOS: 4-6, wherein the sequence encodes aglucanase capable of degrading one or more polysaccharides.40. The method of embodiment 39, wherein the one or more polysaccharidesis selected from the group consisting of beta-glucan, cellulose,cellobiose, pNP-D-glucopyranoside and xylan.41. The method of any or both of embodiments 39-40, wherein expressionof the nucleic acid produces the glucanase and the glucanse is thermallystable upon exposure to a temperature in the range of 25° C. to 130° C.

42. A method for enhancing production of fermentable sugars from grainscomprising:

mixing grains derived from a transgenic plant of any one of any one ormore of embodiments 1-13 with grains derived from a different plant toform mixed grains; and

processing the mixed grains.

43. The method of embodiment 42, wherein the different plant is anengineered plant that includes a synthetic nucleic acid encoding atleast one hydrolytic enzyme.44. The method of any or both of embodiments 42-43, wherein the at leastone hydrolytic enzyme is selected from the group consisting of:xylanase, an endoglucanase, an exoglucanase, cellulase, a feruloylesterase, an intein-modified xylanase, an intein-modified endoglucanase,an intein-modified exoglucanase, an intein-modified cellulase, anintein-modified feruloyl esterase, amylase, phytase and protease.45. The method of any one or more of embodiments 42-43, wherein theprocessing includes at least one operations selected from the groupconsisting of harvesting, baling, grinding, milling, chopping, sizereduction, crushing, pellitizing, extracting a component from the mixedgrains, purifying a component or portion of the mixed grains, extractingor purifying starch, hydrolyzing polysaccharides into oligosaccharidesor monosaccharides, ensiling, fermentation, chemical conversion, andchemical catalysis.46. The method of embodiment 45 further comprising producing abiochemical product.47. The method of embodiment 46, wherein the biochemichal product isselected from the group consisting of ethanol, butanol, lactic acid,citric acid, and acetic acid.48. A method for enhancing ethanol production from grains comprisingperforming a method of any one or more of embodiments 42-47.49. A method for enhancing ethanol production from a transgenic plantcomprising:

mixing a transgenic plant or part thereof of any one or more ofembodiments 1-13 with a different plant or part thereof to form mixedplant material;

converting the mixed plant material into fermentable sugars; and

processing the fermentable sugars into ethanol.

50. The method of embodiment 49, wherein the plant material includesfiber, grain, or a combination thereof.51. A synthetic polypeptide that includes an amino acid sequence with atleast 70% identity to a reference sequence selected from the groupconsisting of: SEQ ID NOS: 4-6, and capable of degrading one or morepolysaccharides.52. The synthetic polypeptide of embodiment 51, wherein the one or morepolysaccharides is selected from the group consisting of beta-glucan,cellulose, cellobiose, pNP-D-glucopyranoside and xylan.53. A synthetic polypeptide that includes an amino acid sequencecomprising a contiguous amino acid sequence having at least 90% identityto 50 to 100, 50 to 150, 50 to 200, 50 to 250, 50 to 300, 50 to 322, or50 to all contiguous amino acid residues of a glucanase having thesequence of any of SEQ ID NOS: 4-6, wherein the glucanase is capable ofdegrading one or more polysaccharides.54. The synthetic polypeptide of embodiment 51, wherein the one or morepolysaccharides is selected from the group consisting of beta-glucan,cellulose, cellobiose, pNP-D-glucopyranoside and xylan.55. A method of increasing metabolizable energy of a diet comprisingmixing a transgenic plant or part thereof with a feed ingredient,wherein the transgenic plant or part thereof comprises a syntheticnucleic acid encoding a glucanase comprising an amino acid sequence withat least 70% identity to a reference sequence selected from the groupconsisting of SEQ ID NOS: 4-6, and and capable of degrading one or morepolysaccharides.56. The synthetic polypeptide of embodiment 55, wherein the one or morepolysaccharides is selected from the group consisting of beta-glucan,cellulose, cellobiose, pNP-D-glucopyranoside and xylan.57. The method of any one or both of embodiments 55-56, wherein thesynthetic nucleic acid comprises a sequence with at least 70% identityto a reference sequence selected from the group consisting of: SEQ IDNOS: 1-3.58. The method of any one or more of embodiments 55-57, wherein theglucanase is active upon expression in the plant and exposure to a pH inthe range from 5.0 to 10.0.59. The method of any one or more of embodiments 55-58, wherein theglucanase is active upon expression in the plant and exposure to atemperature in the range from 25° C. to 130° C.60. The method of any one or more of embodiments 55-59, wherein the feedingredient includes at least one component selected from the groupconsisting of: corn meal, corn pellets, wheat meal, wheat pellets, wheatgrain, wheat middlings, barley grain, barley pellets, soybean meal, soyhulls, dried distillers grain, soybean oilcake, sorghum grain andsorghum pellets.61. The method of any one or more of embodiments 55-60, wherein the feedingredient includes at least one component selected from the groupconsisting of: soluble solids, fat and vermiculite, limestone, plainsalt, DL-methionine, L-lysine, L-threonine, COBAN®, vitamin premix,dicalcium phosphate, selenium premix, choline chloride, sodium chloride,mineral premix, and one or more exogenous enzymes.62. A method for producing an animal feedstock comprising mixing atransgenic plant or part thereof of any one or more of embodiments 1-13with plant material. The method may also comprise the method forproducing a plant that includes a glucanase of any one or more ofembodiments 63-68.63. A method for producing a plant that includes a glucanase comprisingcrossing a plant with a transgenic plant comprising event 4588.259,4588.757 or 4588.652, and selecting a first progeny plant comprisingevent 4588.259, 4588.757 or 4588.652 and capable of degrading one ormore polysaccharides.64. The method of embodiment 63, wherein the one or more polysaccharidesis selected from the group consisting of beta-glucan, cellulose,cellobiose, pNP-D-glucopyranoside and xylan.65. The method of any one or more of embodiments 63-64 furthercomprising selfing the first progeny plant and selecting a secondprogeny plant comprising event 4588.259, 4588.757 or 4588.652 andcapable of degrading one or more polysaccharides.66. The method of embodiment 65, wherein the second progeny plant ishomozygous for event 4588.259, 4588.757 or 4588.652.67. The method of embodiment 65, wherein the second progeny plant isheterozygous for event 4588.259, 4588.757 or 4588.652.68. The method of embodiment 67 further comprising selfing the secondprogeny plant and selecting a third progeny plant homozygous event4588.259, 4588.757 or 4588.652 and capable of degrading one or morepolysaccharides.69. A kit for identifying maize event 4588.259, 4588.757 or 4588.652 ina sample comprising a first primer and a second primer, wherein thefirst primer and the second primer are capable of amplifying a targetsequence specific to maize event 4588.259, 4588.757 or 4588.652.70. The kit of any one or more of embodiment 69 wherein, the firstprimer comprises a nucleic acid sequence selected from SEQ ID NOS: 38,41, and 47.71. The kit of any one or more of embodiments 69-70, wherein the secondprimer comprises a nucleic acid sequence selected from SEQ ID NOS: 39,42, 43, 45, and 46.72. The kit of any one or more of embodiments 69-71, wherein the targetsequence comprises a sequence selected from the group consisting of SEQID NOS: 51-55.73. The kit of any one or more of embodiments 69-72, wherein the targetsequence is capable of hybridizing to the sequence of the nucleic acidcomprising a sequence of SEQ ID NOS: 40 or 44 under conditions of highstringency.74. The kit of any one or more of embodiments 69-73, wherein the samplecomprises plant matter derived from a transgenic plant of any one ormore of embodiments 1-13.75. A method of identifying maize event 4588.259, 4588.757 or 4588.652in a sample comprising:

contacting a sample with a first primer and a second primer of the kitof any one or more of embodiments 69-74;

amplifying a nucleic acid in the sample to obtain an amplified product;and

detecting an amplified product specific to a target sequence in maizeevent 4588.259, 4588.757 or 4588.65.

76. The method of embodiment 75, wherein the target sequence comprises asequence selected from SEQ ID NOS: 51-55. The method of identifying maybe added to any one or more of embodiments 63-68.77. The method of embodiment 75, wherein the target sequence is at leastone sequence selected from the group consisting of SEQ ID NOS: 22-31.78. The method of embodiment 75, wherein the step of detecting compriseshybridizing the amplified product to the nucleic acid comprising asequence of SEQ ID NOS: 40 under conditions of high stringency, andselecting the amplified product specific to maize event 4588.259.79. The method of embodiment 75, wherein the step of detecting compriseshybridizing the amplified product to the nucleic acid comprising asequence of SEQ ID NOS: 44 under conditions of high stringency, andselecting the amplified product specific to maize event 4588.652.80. A method for reducing the viscosity of a grain mixture comprisingcombining grains from a transgenic plant of any one or more ofembodiments 1-13, a different plant, and liquid to form a grain mixture.81. The method of embodiment 80, wherein the different plant is anon-transgenic plant.82. The method of embodiment 80, wherein the different plant is agenetically engineered plant.83. The method of embodiment 80 and 82, wherein the geneticallyengineered plant comprises a synthetic nucleic acid encoding at leastone hydrolytic enzyme.84. The method of embodiment 83, wherein the at least one hydrolyticenzyme is selected from the group consisting of: xylanase, an amylase,an endoglucanase, an exoglucanase, a feruloyl esterase, a glucoamylase,an intein-modified amylase, an intein-modified xylanase, anintein-modified endoglucanase, an intein-modified exoglucanase, anintein-modified feruloyl esterase, a protease, an intein-modifiedprotease, a phytase, or an intein-modified phytase.85. The method of any one or more of embodiments 80-84 furthercomprising processing the grain mixture.86. The method of embodiment 85, wherein the step of processing includesone or more operations selected from the group consisting of harvesting,grinding, milling, size reduction, crushing, heating, gelatinizing,liquefaction, extracting a component from the mixed grains, purifying acomponent or portion of the mixed grains, extracting or purifyingstarch, hydrolyzing polysaccharides into oligosaccharides ormonosaccharides, saccharifying, fermentation, chemical conversion, andchemical catalysis.

Further embodiments herein may be formed by supplementing an embodimentwith one or more element from any one or more other embodiment herein,and/or substituting one or more element from one embodiment with one ormore element from one or more other embodiment herein.

EXAMPLES

The following non-limiting examples are provided to illustrateparticular embodiments. The embodiments throughout may be supplementedwith one or more detail from one or more example below, and/or one ormore element from an embodiment may be substituted with one or moredetail from one or more example below.

Example 1. Feed Glucanase Expression Vectors

A codon optimized nucleotide sequence for expression of the AGR2314 feedglucanase in maize was synthesized. For generating initial planttransformation constructs, single AGR2314 expression cassettes wereassembled in vectors pAG4000 (pAG4258) or pAG4500 (pAG4588, pAG4597, andpAG4708). The vector pAG4000 has been created by replacing the riceubiquitin 3 promoter with the first intron by the maize ubiquitin 1promoter containing its own first intron for driving expression of theselectable marker gene encoding E. coli phosphomannose isomerase (PMI).The vector pAG4500 represents further improvement of pAG4000 andcontains three modifications such as 1) insertion after the first maizeubiquitin intron of a 9 bp sequence (ATCCAGATC) representing the firstthree codons of the ubiquitin monomer with ATG converted into ATC; 2)insertion of the maize Kozak element (TAAACC) after the 9 bp sequenceubiquitin monomer; 3) replacement of the old multiple cloning site (MCS)by a new MCS that was synthesized by PCR and that was designed tocontain multiple sites for several rare cutting enzymes (NotI, PacI,FseI, SwaI, AscI, AsiSI) to facilitate cloning of up to 4-5 expressioncassettes on one T-DNA.

Sequence of the new MCS in pAG4500 (PmeI-KpnI fragment):

(SEQ ID NO: 20) GTTTAAACTGAAGGCGGGAAACGACAACCTGATCATGAGCGGAGAATTAAGGGAGTCACGTTATGACCCCCGCCGATGACGCGGGACAAGCCGTTTTACGTTTGGAACTGACAGAACCGCAACGTTGAAGGAGCCACTCAGCCTAAGCGGCCGCATTGGACTTAATTAAGTGAGGCCGGCCAAGCGTCGATTTAAATGTACCACATGGCGCGCCAACTATCATGCGATCGCTTCATGTCTAACTCGAGTTACTGGTACGTACCAAATCCATGGAATCAAGGTACC.

FIGS. 1-4 illustrate the expression vectors pAG4258, pAG4588, pAG4597,and pAG4708, respectively, carrying a single feed glucanase expressionunit. The vector pAG4258 (FIG. 1; SEQ ID NO 7) has been cloned byassembling an expression cassette that was composed of the maize Glb1promoter fused to the maize codon optimized AGR2314 sequence inKpnI-AvrII sites of pAG4000. The vectors pAG4588 (FIG. 2; SEQ ID NO 8)and pAG4597 (FIG. 3; SEQ ID NO 9) were developed by assembling theircorresponding AGR2314 expression cassettes in KpnI-EcoRI sites ofpAG4500, while the vector pAG4708 (FIG. 4; SEQ ID NO 10) was produced bycloning AGR2314 expression cassette into XmaI-AvrII sites of pAG4500.FIGS. 5-6 illustrate the expression vectors pAG4766 and pAG4767,respectively, carrying two feed glucanase expression units. FIGS. 7-8illustrate the expression vectors pAG4770 and pAG4771, respectively,carrying three feed glucanase expression units. The unique rare cuttingrestriction sites that are available within the MCS of the pAG4500 weresubsequently used in order to develop additional expression constructscontaining either double AGR2314 expression units, such as pAG4766 (FIG.5; SEQ ID NO 11) and pAG4767 (FIG. 6; SEQ ID NO 12), or triple AGR2314expression units, such as pAG4770 (FIG. 7; SEQ ID NO 13) and pAG4771(FIG. 8; SEQ ID NO 14), on the same T-DNA. The constructed vectors forexpression of AGR2314 glucanase in plants are listed in Table 1. E. colistrains carrying the expression vectors were used for conjugation withAgrobacterium and subsequent transformation of maize.

TABLE 1 Description of Sequences SEQ ID Sequence NO ConstructDescription Type 1 AGR2314 maize—optimized protein DNA coding sequence(including C-terminal ER-retention signal “SEKDEL” 2 AGR2414 codingsequence DNA 3 AGR2514 coding sequence DNA 4 AGR2314 Mature proteinsequence Amino acid (including C-terminal ER-retention signal “SEKDEL”)5 AGR2414 protein Amino acid 6 AGR2514 protein Amino acid 7 pAG4258Glb1:mZ27:AGR2314:SEKDEL:NOS DNA 8 pAG4588 Glu1:mZ27:AGR2314:SEKDEL:T35SDNA 9 pAG4597 mZein:mZ27:AGR2314:SEKDEL:T35S DNA 10 pAG4708Ole:mZ27:AGR2314:SEKDEL:NOS DNA 11 pAG4766 Glu1:mZ27:AGR2314:SEKDEL:NOS,DNA Glb1:mZ27:AGR2314:SEKDEL:NOS 12 pAG4767 mZein:mZ27:AGR2314:SEKDEL,DNA Glb1:mZ27:AGR2314:SEKDEL:NOS 13 pAG4770mZein:mZ27:AGR2314:SEKDEL:NOS, DNA Glu1:mZ27:AGR2314:SEKDEL:NOS,Glb1:mZ27:AGR2314:SEKDEL:NOS 14 pAG4771 Glu1:mZ27:AGR2314:SEKDEL:NOS,DNA mZein:mZ27:AGR2314:SEKDEL:NOS, Glb1:mZ27:AGR2314:SEKDEL:NOS 15pAG4257 mZein:mZ27:AGR2514:SEKDEL:NOS DNA 16 pAG4692Glu1:mZ27:AGR2414:SEKDEL:T35S DNA 17 pAG4693mZein:mZ27:AGR2414:SEKDEL:T35S DNA 18 pAG4705Glu1:mZ27:AGR2514:SEKDEL:T35S DNA 19 pAG4706 Ole:mZ27:AGR2514:SEKDEL:NOSDNA

Expression cassettes for related beta glucanases, AGR 2414 and AGR 2514,were prepared using similar strategies, and sequences are provided forthese expression cassettes as they are found in the expression vectorspAQ4257 pAQ4692, pAQ4693, pAQ4705, pAQ4706, pAQ4766, pAQ4257, pAQ4692,pAQ4693, pAQ4705, and pAQ4706.

Example 2. Feed Glucanase Protein Extraction Procedure

Flour was prepared from about 20 transgenic seeds by milling in an Udycyclone mill or knife mill with 0.5 mm or 1 mm screen. About 0.5 ml ofprotein extraction buffer (100 mM sodium phosphate, pH 6.5, 0.01% Tween20) was added to 20 mg flour in a 2 ml tube. In some cases, 2 g, 10 g,or 20 g ground samples was mixed with 10 ml, 50 ml or 100 ml of theextraction buffer in 15 ml tubes or 250 ml bottles. Larger masses andvolumes can be used by scaling these amounts appropriately. Aftervortexing, the tubes were placed on a rotating platform in a 60° C.incubator and rotated for 1 hour for protein extraction. Aftercentrifugation at 16,000×g for 10 min in a tabletop centrifuge, thesupernatant was diluted 20-fold for enzyme assay by adding 20 μlsupernatant to 380 μl protein extraction buffer. In some cases, otherdilution factors were used, as necessary.

Example 3. Feed Glucanase Activity Measurement

Colorimetric Assay. Fifty microliters of the diluted (20-fold to360-fold) protein extract was mixed with 450 μl of 100 mM sodiumphosphate buffer, pH 6.5, 0.01% Tween 20 and 1 tablet of β-glucazymefrom Megazyme (Wicklow Ireland), and then incubated at 80° C. for 1 hourbefore adding 1 ml of 2% Tris base. After centrifugation at 3000×g for10 min, 100 μl of supernatant was transferred to a microplate forabsorbance measurement at 590 nm (A590). The activity was recorded asA590/mg flour after multiplying the dilution factors: A590xAx(500/50)/20mg, where A is protein extraction dilution factor; 500 is the volume(ml) of buffer used for protein extraction; 50 is the volume of proteinextraction (ml) used in the activity test.

Unit Activity Measurement. The assay involves the quantitation ofreducing sugars that are released during a time course digestion of amodel substrate (barley-β-glucan) obtained from Megazyme (Wicklow,Ireland).

Hydrolysis of model substrate. Test 2 ml tubes were labeled with “+”sign, and 5 mg barley-β-glucan substrate (reaction) was added to eachtest tube; no substrate was added to control tubes (control). Fourhundred fifty microliters of 100 mM sodium phosphate buffer, pH 6.5, wasadded to each tube (reactions and controls), and tubes were placed intoa Thermo-shaker with temperature set at 80° C. and shaking speed set at1000 rpm. Tubes were shaken at 1000 rpm at 80° C. for 20 min until thesubstrate was completed dissolved. A tube with 2 ml of diluted grainprotein extract, extracted as described above was placed in theThermo-shaker to be pre-warmed.

Fifty microliters of the pre-warmed sample were added to the control andreaction tubes. Shaking was resumed and a timer was started. After 15minute of shaking at 80° C., 50 μl of each of the reaction and controlsamples were removed and mixed with 10 μl of 0.5N HCl in separatemicroplates. Shaking of the samples was resumed until all samples wereremoved and mixed with acid.

BCA quantification of glucose reducing equivalents. Glucose standardswere prepared in protein extraction buffer at the followingconcentrations: 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM. BCAreagent (from Thermo Scientific) was prepared by mixing reagent A withreagent B in a ratio of 50:1. To make a glucose standard curve, 75 μl ofbuffer were dispensed into the first well of row A (A1) in a microplateand 75 μl of each glucose standard were dispensed into wells A2 throughA7. To detect the reducing sugars from the feed glucanase reaction andcontrol samples, 25 μl from each reaction were dispensed into rows ofthe microplate in the order of their incubation time withbarley-β-glucan (e.g., row B1-B2: 15 min-30 min), then added 25 μl ofcorresponding control to another row of the microplate (e.g., row C1-C2:15 min-30 min). Subsequently, 50 μl of sodium phosphate buffer weredispensed in each well in these two rows (reaction and control), and 175μl BCA reagent were added to each well using a multichannel pipette.Mixing was achieved by pipetting up and down. The microplate was sealedand incubated at 80° C. in a heat block. After 10 min incubation, themicroplate was chilled on ice for 10 minutes and centrifuged to bringdown condensate. Subsequently, the absorbance at 560 nm of each well wasmeasured on a microplate reader.

Calculating units of feed glucanase activity from A560. The absorbancefrom the reagent blank was subtracted from the absorbance values foreach of the glucose standards, and the resulting values were plottedaccording to their glucose concentrations. Linear regression was thenused to calculate the “best fit” line through the data set. To determineglucose reducing equivalents in glucanase/barley-β-glucan reactions, foreach time point, the absorbance value from the control sample wassubtracted from the reaction sample, and the resulting value was used tocalculate the concentration of reducing sugars by comparison to theglucose standard curve. One unit (U) of glucanase activity is the amountof enzyme required to release 1 μmol glucose reducing equivalents from1% Barley-β-glucan per minute at 80° C., pH 5.3, using the BCA method ofquantitation.

Unit Activity Measurement (semi-high throughput method). As describedherein, the method detects the reducing sugars such as glucose releasedfrom the model substrate (barley-β-glucan) by glucanase treatment at 80°C. for 40 minutes or 90 minutes. When protein extract from grain or feedis appropriately diluted, the initial velocity is detected within 40minutes (grain product) or 90 minutes (feed sample) of the reaction. Thereactions were carried out in 96-well block (Costar, Cat #3960) or striptubes (VWR, Cat #29442-610).

Substrate preparation: Barley-β-glucan (low viscosity) was weighed basedon the number of reactions, e.g., 10 samples, 4 dilutions for eachsample needed a total of 40 reactions. Each reaction needed 5 mgsubstrate, therefore, at least 40×5=200 mg of barley-β-glucan wasrequired. The substrate was completely dissolved with the extractionbuffer at 80° C. water bath for 20 minutes, and vortexed at every 5 to10 minutes.

The cluster tubes were used for 90 minutes endpoint activity unit assayof feed samples. Protein extract was diluted to 2-, 6-, 10- and 20-folddilutions.

Purified protein diluted 100-fold was used as a positive control forassay validation. Purified glucanase protein (200,000 ppb) was stored in50 mM MES, 150 mM sodium chloride, pH6.3 buffer plus 40% glycerol at−20° C. Ten microliters of protein were mixed with 990 μl of theextraction buffer, and 50 μl were used for activity assay.

Barley-β-glucan digestion by feed glucanase was carried out at a waterbath set at 80° C. In the block of cluster tubes, 450 μl of thesubstrate were dispensed into tubes of A2 to D12 referring Table 2.These rows served as the reaction.

Four hundred fifty microliters of the extraction buffer (no substrate)were added to each control tubes from rows E2 to H12, which served asblank to correct protein content detected by BCA method for eachreaction as described in Table 2 (A2 to D12).

Fifty microliters of the diluted sample extract including the negativecontrol and positive control were added first to each blank tube, E2 toH12, and then to each reaction tube, A2 to D12, as described in Table 2.

TABLE 2 Example of enzyme hydrolysis of feed samples in cluster tubes 12 Columns 1 to 11 12 A Neg. Ctr, Sample_X, 2x dilution Pos. Ctr Reaction2x B Neg. Ctr, Sample_X, 6x dilution Pos. Ctr 6x C Neg. Ctr, Sample_X,10x dilution Pos. Ctr 10x D Neg. Ctr, Sample_X, 20x dilution Pos. Ctr20x E Neg. Ctr, Sample_X, 2x dilution Pos. Ctr Blank 2x F Neg. Ctr,Sample_X, 6x dilution Pos. Ctr 6x G Neg. Ctr, Sample_X, 10x dilutionPos. Ctr 10x H Neg. Ctr, Sample_X, 20x dilution Pos. Ctr 20x

The tubes were covered with Corning™ Storage Mat III, the CorningStorage Mat Applicator was used to seal the tubes. The plate was shakenat a low speed. The block was placed in the water bath at 80° C. for the90 minutes incubation period. The reaction was terminated by adding 100μl of 0.5 N HCl to each well and cooling the block on ice.

BCA quantification of glucose reducing equivalents. Glucose standardswere prepared in 100 mM sodium phosphate buffer, pH6, at the followingconcentrations: 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM. BCAreagent (from Thermo Scientific) was prepared by mixing reagent A withreagent B in a ratio of 50:1. To make a glucose standard curve in column1 on a microplate, 75 μl of buffer was dispensed into the first well ofrow A (A1) and 75 μl of each glucose standard were dispensed into wellsA2 through A7. To detect the reducing sugars from the feed glucanasereaction and control samples, 25 μl from each reaction were dispensedinto rows of the microplate according to the order displayed on Table 2.Subsequently, 50 μl of the extraction buffer was dispensed in eachsample well (reaction and blank) referring to Table 2 from A2 to H12 tomake a total volume 75 μl. One hundred seventy five microliters of theBCA reagent were added to each well and mixed. The microplate was sealedand incubated at 80° C. on a heat block. After 10 min incubation, themicroplate was chilled on ice for 10 min and centrifuged to bring downcondensate. Subsequently, the absorbance at 560 nm of each well wasmeasured on a microplate reader.

Calculating units of feed glucanase activity from A560. The absorbancevalue for the reagent blank was subtracted from the absorbance valuesfor each of the glucose standards, and the resulting values were plottedaccording to their glucose concentrations. Linear regression was thenused to calculate the “best fit” line for the data set. To determineglucose reducing equivalents in the glucanase/barley-β-glucan reactions,the absorbance value from the control sample was subtracted fromabsorbance value of the corresponding reaction sample, and the resultingvalue was used to calculate the concentration of reducing sugars bycomparison to the glucose standard curve. One unit (U) of glucanaseactivity equals 1 μmol glucose reducing equivalents released from 1%barley-β-glucan per minute at 80° C., using the BCA method ofquantitation.

Calculating units of positive controls from A560 to validate the assay.The value of absorbance for blank samples (E12, F12, G12, H12) wassubtracted from the value of absorbance for each reaction sample (A12,B12, C12, D12). The regression equation for the glucose standard wasused to calculate the glucose content (μmol). To determine the amount ofreducing units produced per minute (A), the value for the amount ofglucose (μmol) released from barley-β-glucan in the reaction was dividedby the reaction time, for example 90, if the reaction time was 90minutes. The unit value of positive controls equals the dilution x(A)/mg of protein in the assay. The dilution factor in the assaydescribed herein equals 24. The dilution factor of 24 was determined bycomparing the ratio of the total reaction volume to the portion of thereaction that was used in the BCA assay. In the assay, the totalreaction volume was 600 μl including 500 μl reaction and 100 μl of HClused to stop the reaction. The portion of the reaction that was used inthe BCA assay was 25 μl. Therefore, the dilution factor of 24 wascalculated by dividing 600 μl by 25 μl.

The amount of protein in the assay was calculated as follows. Theconcentration of the positive control was 2000 ng/ml, 50 μl was thealiquot of the positive control used in the test (or 50/1000 ifcalculated in mL). The amount of protein calculated in nanograms was2000×(50/1000), or 2000×(50/1000)/1000000 if calculated in milligrams.

Example 4. Glucanase Activity in Seed from Transgenic Maize

Silks on untransformed (wild type) maize plants were pollinated withpollen from individual transgenic maize plants that carried the pAG4588construct. Mature, dried seeds were harvested from the resulting earsand assayed for activity via the colorimetric assay. FIG. 9 illustratesthe range of activities recovered from 42 independent ears. In thisfigure, the numbers along the abscissa correspond to individual eventidentifiers. The highest activity was observed in the event 259. In theT0 transgenic maize plants 757 that also carried the pAG4588 construct,the activity was about 25 A590/mg. The average activity of thehomozygous seeds derived from the first generation of the selfed plantswas approximately 116 f 15 A590/mg. The activity of heterozygous seedsfrom this population was about 59 f 18 A590/mg.

Silks on untransformed (wild type) maize plants were pollinated withpollen from individual transgenic maize plants that carried the pAG4597construct. Mature, dried seed were harvested from the resulting ears andassayed for activity via the colorimetric assay. FIG. 10 illustrates therange of activities recovered from 15 independent ears. In this figure,the numbers along the abscissa correspond to individual eventidentifiers. The highest activity was observed in the event 460.

Example 5. Maize Genomic Sequences Flanking T-DNA Integration Sites inTransgenic Events 4588.259, 4588.757 and 4588.652

Event 4588.259: The event 4588.259 carries two independent T-DNAintegration sites that are located on the maize chromosomes 4 and 8. Thechromosomal locations of the T-DNA integration sites were determinedthrough BLASTN searches, in which the maize genomic DNA sequences thatare contained in OB-2880, OB-2832 and OB-3252 sequences isolated fromT-DNA insertion sites at the right and left T-DNA borders, were used asthe queries for screening publicly available maize B73 genome sequencedatabases, such as the Maize Genetics and Genome Database,htt)://www.maizegdb.org/(Accessed May 8, 2016) See also Andorf, C M,Cannon, E K, Portwood, J L, Gardiner, J M, Harper, L C, Schaeffer, M L,Braun, B L, Campbell, D A, Vinnakota, A G, Sribalusu, V V, Huerta, M,Cho, K T, Wimalanathan, K, Richter, J D, Mauch, E D, Rao, B S, Birkett,S M, Richter, J D, Sen, T Z, Lawrence, C. J. (2015) MaizeGDB 2015: Newtools, data, and interface for the maize model organism database.Nucleic Acids Research doi: 10.1093/nar/gkv1007; Lawrence, C J,Seigffried, T E, and Brendel, V. (2005) The Maize Genetics and GenomicsDatabase. The community resource for access to diverse maize data. PlantPhysiology 138:55-58; Lawrence, C J, Dong, Q, Polacco, M L, Seigfried, TE, and Brendel, V. (2004) Maize GDB, the community database for maizegenetics and genomics. Nucleic Acids Research 32:D393-D397, all of whichincorporated herein by reference as if fully set forth. Because bothloci segregate independently, plants carrying both loci and eachindividual locus were evaluated.

In the flanks OB-2880, OB-2832 and OB-3252, which are provided below,the maize genomic DNA is shown in the uppercase letters, while thepAG4588 vector sequences are indicated in the lowercase letters and areunderlined.

Integration Site on the Maize Chromosome 4:

The T-DNA integration site on the maize chromosome 4 is characterized bythe 795 bp right T-DNA border flanking sequence OB-2880, which contains677 bp of maize genomic. The isolated 677 bp maize genomic DNA flank has99.3% sequence identity to the sequence derived from the antisense DNAstrand of the maize chromosome 4 (nucleotide coordinates56612593-56612026).

>OB-2880 (SEQ ID NO: 22)CTTAGATTAGAGAATGAAAATTTGATTGCTAAGGCCCAAGATTTTGATGTTTGCAAAGATACAATTACCGATCTTAGAGATAAGAATGATATACTTCGTGCTAAGATTGTTGAACTTACACCACAACCTTCTATGCCTTCTGTGACATTAACATTACGTCACAAACAATAGTATTTTTGTCATACCTTACATGTTGGTGACGTGATTGTGACGAAAATCACATCGTCACAGAAGGTGCGTGTTAAATGGTGTACTATGACGAATAACAAAAAAACGTCATAATAGTTTATGACGCAAACTACAAACGTCACTAATCTATGACACTCGAATTCGTCACTAATTATGTCTAAATACGTCACAATTCATGTAGTCGTGCCTTGCCACGTGGCTGATTACGTGGCGAGATGACATGGCAGTTGACGTGGCAGGTGATGTGGCGAAAATGTTGTGACGAGTTCATTCGTCACAGATGTTATGACGTGGCATGCCACATGGCAGATGATGTGGCAAAATTATGTGACAAAAATATTTGTCATAAATATCAATGAGGTGGCAATATATGTGTGACGAAATTTTTCATCACAAAGTACGATGACGTTGCAATATATTTATGACGAATTGTTCATCATAAGGCGTGATGAATTCATAGCGTCATGGAATATTATGAAATCACATGCtcaaacactgatagtttaaactgaaggcgggaaacgacaacctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcgggacaagccgttttacgtttgg

Integration site on the maize chromosome 8:

The T-DNA insertion site on the chromosome 8 is characterized by thesequences OB-2832 and OB-3252 that represent, accordingly, left andright flanks for the T-DNA integrated into this locus.

The 1211 bp OB-2832 sequence contains 864 bp of maize genomic DNA. Theisolated 864 bp maize genomic DNA flank has 99.65% sequence identity tothe sequence derived from the maize chromosome 8 with nucleotidecoordinates 100613054-100613915.

>OB-2832 (SEQ ID NO: 23)tcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattggcgagctcgaattaattcagtacattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatACTAAAAAAACTCAAGGATCTGTCTCCAGAAAGGCCTTGCAGGGTTTGGCCACGCCCACGGACATTCCATCTCAGAGCCATGATTAGAACGAAAAACACATGAGAGCCGTCGTTGCTAGGAGTCGGTTTCATATGTTCGCTAAAACAAGAGATTTGTTTTTTTTCTCTCTCGTACATACACGAGTCAGCCCTTTTAATCTCAGGTTGACGTGCAATGTCGCTCGTCTAAGCAGAACATTTTGAGAACAAATGTGTTGTACATGAGAGTTTTGTGTACATGGTACGTACATTAAAACATCATCATTTATCTTAGATCTAACATCTCTACTTGCTTGTTATATATTTTTTTTGTAAAATAACATCTTTCACCACTTTATATGGTGTTGTTTGCAAAATATACAGAGCAATTAGAGACGTTAGATTTGAGATGGACGGTGATAATTTAATACATGCATAATGTACAAGAAAATCCTAACTGCACTAGATATGTTGTCAAACATTTTACCTTTGTTACAAAAAGAAATGAATAGATGTTGAACGGTTGTCTTTCAAGCCTGTTCGCTGCGGCTTTAATTCACCAACTGCAATGAACAACCTGAAAGGTGATCGTTGCCGAACACATGCTGTTTGGCAAAGCTAGTAGTACCTTTTTTGTCTGTCACCTGGAATGATGAGAAAGGAGACAAGAGGAGAGGGCTGGCCATTGTTTATATATATACGTATTTCCATTGCTTTGTGGCATGCAACAGTTCAAGGGTCCAAACTGGCAGGTTTTCAGCCCCGACAAATATAATAAAAAAACTACAAAAAAAAAAGGTCCGTTTACATTCCTTTTTTGACAACGCTAGTCCGT GCGGAGCGAGC

The 696 bp OB-3252 sequence contains 95 bp of maize genomic DNA. TheOB-3252 does not contain left T-DNA border sequence. The isolated 95 bpflank has 100% sequence identity to the sequence derived from theantisense DNA strand of the maize chromosome 8 with nucleotidecoordinates 100613034-100612940.

>OB-3252 (SEQ ID NO: 24)GgtgaaacaaggtgcagaactggacttcccgattccagtggatgattttgccttctcgctgcatgaccttagtgataaagaaaccaccattagccagcagagtgccgccattttgttctgcgtcgaaggcgatgcaacgttgtggaaaggttctcagcagttacagcttaaaccgggtgaatcagcgtttattgccgccaacgaatcaccggtgactgtcaaaggccacggccgtttagcgcgtgtttacaacaagctgtaagagcttactgaaaaaattaacatctcttgctaagctgggagctctagatccccgaatttccccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattggcgagctcgaattaaTTCAAGTGTCTTCGTACAAACTGGGGGATGGGGCAGACCGCCAGGTTCAAACCGTTTGACTAGATGCGGCTGGCAGGCTACTTTGCAGTGCATGC

The maize genomic DNA flanks in sequences OB-2832 and OB-3252 areseparated by 20 nucleotides on the maize chromosome 8, which indicatesthat during T-DNA integration 20 bp of the original maize genomic DNAsequence were replaced by the inserted T-DNA sequences.

There is also an OB-2861 sequence and an OB-2868 sequence within the 259event. The 970 bp OB-2861 sequence consists of the re-arranged pAG4588sequences including a partial 223 bp Nos terminator sequence (uppercaseletters, nucleotides 3290-3512 in pAG4588); the 73 bp sequence near theleft T-DNA border with the first 3 bp of the processed left T-DNA bordersequence (italicized lowercase letters, nucleotides 3513-3585); the 299bp sequence near the right T-DNA border with 5 bp of the processed rightT-DNA border sequence and polylinker sequence with multiple cloningsites (lowercase letters, nucleotides 9647-9945); the 359 bp 5′ sequenceof the rice glutelin promoter prGTL-03 (uppercase letters, nucleotides9946-10304). The underlined are 18 bp of a duplicated sequence that hasbeen created during T-DNA integration process. The OB-2861 sequence isas follows:

(SEQ ID NO: 25) GAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTGgcgagctcgaattaattcagtacattaaaaacgtccgcaatgtgttatt aagttgtctaagcgtcaa tttgttatcaagttgtctaagcgtcaaacactgatagtttaaactgaaggcgggaaacgacaacctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcgggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagcctaagcggccgcattggacttaattaagtgaggccggccaagcgtcgatttaaatgtaccacatggcgcgccaactatcatgcgatcgcttcatgtctaactcgagttactggtacgtaccaaatccatggaatcaaggtaccTCCATGCTGTCCTACTACTTGCTTCATCCCCTTCTACATTTTGTTCTGGTTTTTGGCCTGCATTTCGGATCATGATGTATGTGATTTCCAATCTGCTGCAATATaAATGGAGACTCTGTGCTAACCATCAACAACATGAAATGCTTATGAGGCCTTTGCTGAGCAGCCAATCTTGCCTGTGTTTATGTCTTCACAGGCCGAATTCCTCTGTTTTGTTTTTCACCCTCAATATTTGGAAACATTTATCTAGGTTGTTTGTGTCCAGGCCTATAAATCATACATGATGTTGTCGTATTGGATGTGAATGTGGTGGCGTGTTCAGTGCCTTGGaTTTGAGTT TGATGAGAGTTGCTTCTGGG

The 1127 bp OB-2868 sequence consists of re-arranged pAG4588 sequencesincluding the 595 bp 3′ sequence of the PMI marker gene (uppercaseletters, nucleotides 2594-3188 in pAG4588); the 48 bp sequence betweenPMI and Nos terminator (lowercase letters, nucleotides 3189-3236); the276 bp Nos terminator sequence (uppercase letters, nucleotides3237-3512); the 73 bp sequence near the left T-DNA border with the first3 bp of the processed left T-DNA border sequence (italicized lowercaseletters, nucleotides 3513-3585); the 119 bp sequence near the rightT-DNA border with 5 bp of the processed right T-DNA border and a partialpolylinker sequence (lowercase letters, nucleotides 9647-9765). Theunderlined are 18 bp of a duplicated sequence that has been createdduring T-DNA integration process. The OB-2868 sequence is as follows:

(SEQ ID NO: 26) AAAATCCCGCGCGCTGGCGATTTTAAAATCGGCCCTCGATAGCCAGCAGGGTGAACCGTGGCAAACGATTCGTTTAATTTCTGAATTTTACCCGGAAGACAGCGGTCTGTTCTCCCCGCTATTGCTGAATGTGGTGAAATTGAACCCTGGCGAAGCGATGTTCCTGTTCGCTGAAACACCGCACGCTTACCTGCAAGGCGTGGCGCTGGAAGTGATGGCAAACTCCGATAACGTGCTGCGTGCGGGTCTGACGCCTAAATACATTGATATTCCGGAACTGGTTGCCAATGTGAAATTCGAAGCCAAACCGGCTAACCAGTTGTTGACCCAGCCGGTGAAACAAGGTGCAGAACTGGACTTCCCGATTCCAGTGGATGATTTTGCCTTCTCGCTGCATGACCTTAGTGATAAAGAAACCACCATTAGCCAGCAGAGTGCCGCCATTTTGTTCTGCGTCGAAGGCGATGCAACGTTGTGGAAAGGTTCTCAGCAGTTACAGCTCAAACCGGGTGAATCAGCGTTTATTGCCGCCAACGAATCACCGGTGACTGTCAAAGGCCACGGCCGTTTAGCGCGTGTTTACAACAAGCTGTAAgagcttactgaaaaaattaacatctcttgctaagctgggagctctagaTCCCCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGAATTGgcgagctcgaattaattcagtacattaaaaa cgtccgcaatgtgttattaagttgtctaagcgtcaa tttgttatcaagttgtctaagcgtcaaacactgatagtttaaactgaaggcgggaaacgacaacctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcgggacaagccgttttacgtttgg

Event 4588.757: The event 4588.757 carries one T-DNA integration sitethat is located on the maize chromosome 8.

The chromosomal location of the T-DNA integration site was determinedthrough BLASTN searches, in which the maize genomic DNA sequences thatare contained in OB-3170 and OB-3237 sequences isolated accordingly fromT-DNA insertion sites at the right or left T-DNA borders, were used asthe queries for screening publicly available maize B73 genome sequencedatabases, such as http://www.maizegdb.org/. See also Andorf, C M et al.(2015) Nucleic Acids Research doi: 0.10.1093/nar/gkv1007; Lawrence, C Jet al, (2005) Plant Physiolgy 138:55-58; Lawrence, C J et al., (2004)Nucleic Acids Research 32:D393-D397, all of which incorporated herein byreference as if fully set forth.

In the flanks OB-3170 and OB-3237, which are provided below, the maizegenomic DNA is shown in the uppercase letters, while the pAG4588 vectoris indicated in the lowercase underlined letters.

The 1303 bp OB-3170 right T-DNA border flanking sequence consists of the975 bp maize genomic DNA attached to the 328 bp of the pAG4588 vectorborderless sequence proximal to the right T-DNA border site. Theisolated 975 bp maize genomic DNA flank has 99.38% sequence identity tothe sequence derived from the maize chromosome 8 with nucleotidecoordinates 62661042-62662016.

The OB-3170 sequence is as follows:

(SEQ ID NO: 27) TTGGGGTTCCTTATCCTGTTGTCGGAGTTGTGCCATTATCCTTTCCATGGTTGACCTGAGCTTTAGCCTGTACACTGTAGACTCTACTAGAGGTTTACCTGAGGCTGAATTCCCGCTGCTAAGATGTGATGTTCCCGGCCATAAGCAAAGATGCAGGTTGTCTTTGCTTTGTAAAGATGAAGGTTGTCTTTGTTTTGTAATCGAAAAAAAAACCCTCCGACTTCGATAGCAATCCATTTCTTGAAACGATATAGCTATAAGCTGCAGCCACACCTTGCGTTGATGATGCCAAAGCTTTCTTTCGAGTGCGATGCATGCACTGGCCTGTTGAGATCTTATCAATATGGCAAACAGTAACCTAACGTATATGACTACATGGTCTTCATGCTTTTGAGAGGTGCCTCATAGGAAACAGTCAGGCCAATGATTTTAGGGAATACAATATATTTTTGCTGTTTTTTTTTTGCAAATTGTCCATATTATTACAAAAAAAACTAAACATGCCCAAAGGCAATAGCTTTCTAAATAAAAATGAATAACGGTCCACTTATATATGTTGGCCAGTAATCAATTCTGAGGCCTGACAAACCATGCATATATTAACAGTAGGTTAATGGCCGTGCGTGAAAAAATTTCAATACAACAAGAGATTGAAAAAAAAGAGTGTCTTACCAATATGTTATTTTATAAGTACCAAATGTGTAGGAAACTTGCATTCATTTTTTCCCTGAGAATGGAAAAAAACAAGACATACTCATTTTCAAGTTGAATTGTCATAGCAACACACATGTTGTATCTGCCGGTTCATGCAATTGTGCCAACCAAAATATCTAAATGAGATATTCAAGACTCAACAGAATTAAAGTATGGAATAGGGTGTATATACACTCAACCATTATTAAATGGTATAATCATCTATCTATATCACTATAAAATCTACCAGTTTAAACTTCACAAAACTCATCTAGCTAATGGaggcgggaaacgacaacctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcgggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagcctaagcggccgcattggacttaattaagtgaggccggccaagcgtcgatttaaatgtaccacatggcgcgccaactatcatgcgatcgcttcatgtctaactcgagttactggtacgtaccaaatccatggaatcaaggtacctccatgctgtcctactacttgcttcatccccttctacattttgttctggtt ttg

The 960 bp OB-3237 left T-DNA border flanking sequence consists of the620 bp of the maize genomic DNA attached to the 340 bp pAG4588 vectorsequence (nucleotides 3260-3599 in pAG4588), which includes 253 bp Nosterminator sequence and 70 bp sequence upstream of the 17 bp processedleft T-DNA border sequence. The isolated 620 bp maize genomic DNA flankhas 100% sequence identity to the sequence derived from the maizechromosome 8 with nucleotide coordinates 62662037-62662642.

The OB-3237 sequence is as follows:

(SEQ ID NO: 28) AaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgggaattggcgagctcgaattaattcagtacattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatataAAATCTACCTGTTCGCTGATAAGCCGTTAGGTTGACTATGTGACTGTTGGGCGGCAAAATGACCACGCGGACGGTCTAGCCCCAAAGCCGGACGGTCCGCGGTCCAGACAGTCTGCACTGGTGGTGTCGGCGTTTCGACCCCGGGGGGTCCCTGGACCGACGAGTAAATTGTCGCTGCGTGTCCCAGCCCAGATGGGTCCGCGCGAGACGGAACGCGAAGATGGGAAAACAGCAAAGGGGAACCCGCGGCCTTCGTGTTGTCCTGCGCCCAGGTCGGGTGCGCTTGCAGTAGGGGGTTACAACCGTTCGCGTGGGAGAGACAGAGAGAGAGCGAGAGCCTTATGCGTCGGCCCGTTCTCCCGCGCGGCCAACCCTCTCGTACGAGAGCCCTGGACCTTCCTTTTATAGACGTAAGGAGAGGGCCCAGGTGTACAATGGGGGGTGTAGCAGAGTGCTAACGTGTCTAGCAGAGAGGAGCCGGAGCCCTAAGTACATGTCGTCGTGGCTGTCGGAGAGGTTTTGGCGCCCTGTTCATGTGATGTCGTGGCCGTCGGAGGAGCGCTTGAGCCCCGTGGAAGTACAGCTGTCGGGGCTGTCGGATCCTTGCTGA CGTCTCCTTG

The maize genomic DNA flanks in sequences OB-3170 and OB-3237 areseparated by 21 nucleotides on the maize chromosome 8, which indicatesthat during T-DNA integration 21 bp of the original maize genomic DNAsequence were replaced by the inserted T-DNA sequences.

Event 4588.652: FIG. 11 illustrates a diagram showing positions of thecharacterized flanking sequences in 4588.652.

Sequences isolated at the T-DNA insertion site in 4588.652. T-DNA in theevent 4588.652 has integrated into chromosome 7 of the maize genome inBxA genotype, which was used for maize transformation with the pAG4588construct. The T-DNA insertion occurred between nucleotides141683320-141683357 of the publicly available reference B73 maizegenome. The T-DNA integration displaced 38 bp of the native maizegenomic sequence at this site. This 38 bp DNA is underlined and thesequences of pAG4588 are underlined and shown in bold characters in thesequences of the T-DNA insertions shown below. The diagram illustratedin FIG. 11 depicts locations of the sequences in the locus in 4588.652.The right and the left border T-DNA flanking sequences, OB-4448 andOB-4451 respectively, were isolated from multiple 4588.652 progeny usinga PCR-based genome walking approach. The entire genomic regions betweenthe right and the left border flanks were isolated and sequencecharacterized from WT genotypes BxA, 19545 (E), 15009 (G) as well as thenulls BC2ES2_512x and BC1GS2_518x. The following wild type maize genomicDNA sequences were used for reference: the WT_BxA (OB-4541; SEQ ID NO:32), the WT_E sequence (OB-4545, OB-4546; SEQ ID NO: 33), the WT_Gsequence (OB-4547, OB-4548; SEQ ID NO: 34), the Null_BC2ES2_512xsequence (OB-4578 to OB-4580; SEQ ID NO: 35), the Null_BC1GS2_518xsequence (OB-4582 to OB-4584; SEQ ID NO: 36), and theWT_B73Chr7_141681606-141685147 reference sequence (SEQ ID NO: 37).Furthermore, the right and the left border flanks were additionallyisolated and entirely sequenced from the more advanced 4588_652transgenic progeny BC2ES2_472x. The entire 4044 bp BxA genomic sequencecontaining the right and the left border flanking sequences have highBLASTN identity hits to two nucleotide positions 141681606-141682538 and141682550-141685147 on the chromosome 7 in the maize B73 genome.

Analysis of Nucleotide Sequences in the Left T-DNA Border Flank

The left T-DNA border flank OB-4451 has 98.66% BLASTN sequence identityto nucleotides 141683358-141685147 on the maize chromosome 7 in B73genome. Multiple sequence alignment of the left border specificsequences from the wild type genotypes B73, BxA, 19545 (E), 15009 (G),nulls BC2ES2_512x and BC1GS2_518x as well as 4588_652 transgenicprogenies 116_F1G and BC2ES2_472x and the reference public sequence ofB73 genome revealed that these 1.8 kb sequences are nearly 100%identical between all genotypes.

Analysis of Nucleotide Sequences in the Right T-DNA Border Flank

The 2218 bp right border flank OB-4448 has high BLASTN sequence identityto two nucleotide positions 141681606-141682538 and 141682550-141683319on the maize chromosome 7 in B73 genome. Multiple sequence alignment ofthe right border specific sequences from 4588_652 transgenic progenies116_F1G and BC2ES2_472x with the WT sequence of BxA revealed that thesethree 2.2 kb sequences are nearly 100% identical.

A 521 bp “unique” sequence that is specific to the right T-DNA borderflank has originated from genotype BxA, which was used fortransformation with the pAG4588 construct. No BLASTN sequence identityhits to this sequence were identified at the T-DNA integration sitewithin the reference B73 genome. On the other hand, the 521 bp sequencehas multiple BLASTN identity hits on different chromosomes in maize B73genome indicating that this sequence is highly repetitive. The 521 bpsequence is shown in italicized lowercase letters.

Sequences characterized at 4588_652 T-DNA integration site. The OB-4448sequence (extended right border flank in 4588_652 isolated from F1G of4588_652 is as follows:

(SEQ ID NO: 29) CACCCTCGCTGTTGGTAAACGTGCGCCTTGGGTATGTCCTCACCTGCATGATACGACATGTTGAAAAAGGTACATGGCTGGGCGGATTTAAACAGTAGAATGAAAAGGTGCCACAAGAAAACTCGTCAAAGAATTGACTACGCGTCAATGTTCCATAGTTAAAAAGACTTGAACTCTGGATCAGGGACTTTCAAACAAGGATAGCTGCCTGGTCACCAGTCATTAACTGTAATGTAATGGCCATAGATGATGCATGAGTACAATAATAAAAAAACACCATCCAGCCAAATATATACTCCCTGTCACAAATGAAAATTCGTTTTAGATAATTAGTGGATTCATACAATATTTGTTGTATGTGTTTTATGTGTCTAGATTCATCATCCTCTATTTGAATATAGACAGAAAAATCATAACTAAAACGAATACTATTTGGGAACGGAGGGAGTACTACTTTGGCAGAATGCCCCCAGGAAAGTACCAGTTTCAGGGGTAGTTTGGAAGGCTAAACCTAGGGAGGGAAAACCCCCCACATGTAACTAAATATCTTATTCAAATGTTACCCCTAGGGATTACTCACCCTGGGAAATGAGAAGGGTCCCAAGGGGATTTCGGTTTCTATTATTTTTTCTGCAAACCATTTCAGAGCAATGATATGAAACCAAGCTAACTACTTATAACATTTCTTAAGAATATCAGACATAGGAAAGTGATGGCCTGGAACCAAAGTAAGACTGGTAGATAAATAGATCACTAGAATAAACCCTGACAGTTCATAGCCTTCATAGAAGCAAAAGGAAACACTACGGGAGCAATTGGTTGCTTGCACTAGCAATTCACTGCATTGGGTCTAATGCAGGATAGACTAAGCCAGCATAAGTGTGCGCAATGTGTTTGTGTTTGGTTGCCATGTTATAAGTAAGTTGCATTTGCTAATATctttctcctgactctaatgagtccacttttgctgactggtgggcgaaagtaagtaagcaagtgcacaaatccaaaagaagaggctttaacagtatcatcatcttgggggcttggtgtttatggcttcatcgtaataaggtggtttttgatggtgtcagtccttcaattattggcataaaggcaatttttttggatgaagttgaattctggaggcttgccggtgctaggcatcttgaggctttggttcctggtgctggaatttttaggtcaagggttcttttgggtgattagtgaagagcaggtgtgtgtggtctgctcgcactttttgttgttcgttctcctattgcgtgctgttgtttccaggcgcatttatggaggctgcagttttgtgcgcagcagaagttggtggttttgtgttttgtgttttgcctattttggcattgtactttggtccattttggactgttttcttctcttaatttaatgatgtgcagctctcctgcgcgtttaagaaaaaaaaaAGTTGGCTGTTTTGTATTTCTTGTGATCACCCATGCTTGTTGTGGTCAGATTAAACTCTCACGTTTAATGCTACAGAAGCATCCATGAGACAATGAAACACCGCTCAAAAGCCACGTAGTAGCATACCCTGACTTATGAATAAAGCAACTCGATCTGATTTATTTGAGAAAACAGGAAACTGACAAGTTATTTTTAACACAAAATTTCATTAAAAACGAATGGTAGACAATTACCAATCTGTAGGTCCCTGGCTTGCAAGTCCTCCCAATGTCTAAGAAATCAAATAGGAACTGCAGGCAAGCCAGCAAGAAAGTATTAATCACTGGATATAAAATATAAAGAAAAAAGAAGGAAAGACGGCTACTCGGCTAGCATATGTTTTTGTTAGGGGTGAAAATGGATACTTATTCAGAAATCATTTTTGATCTTTTTTCTTTAATTAGGAATAAATAGGATATAGAATATGCTAAGCAAATTCATATTCTTGTTCTTAGCATTGGGCTTGTAAAGATTCATAAAAGGTAAATCTCAAATTTATCATATATCTTAAATGGTAGATATAAAATTCAGATACAAATATTTTTCAACTTTTTTGTTGTAGGGAACAAATTATATTAAAAAAAATTATGCACAATTCTATTCTTATTTGTAATAATGTGCTTGATAACATAATAAAAGATTACCATCAAATTTCACACACACCCACCCACCCACCCACCCCTGCACGCACGCGCGCGCACACACACTATATGTGTGTtcaaacactgatagtttaaactgaaggcgggaaacgacaacctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcgggacaagccgttttacgtttggaactgacagaaccgcaacgttgaaggagccactcagcctaagcggccgcattggacttaattaagtgaggccggccaagcgtcgatttaaatgtaccacatggcgcgccaactatcatgcgatcgcttcatgtctaactcgagttactggtacgtaccaaatccatggaatcaaggtacctccatgctgtcctactacttgcttcatccccttctacattttgttctggtttttggcctgcatttcggatcatgatgtatgtgatttccaatctgctgcaatatgaatggagactctgtgctaaccatcaacaacatgaaatgcttatgaggcctttgctgagcagccaatcttgcctgtgtttatgtcttcacaggccgaattcctctgttttgtttttcaccctcaatatttggaaacatttatctaggttgttttgtgtccaggcctataaatcataaatgatgttgtcgtattggatgtgaatgtggtggcgtgttcagtgccttggatt tgagt

The RB_BC2ES2_472x sequence (extended RB flank isolated from theadvanced progeny of 4588_652) is as follows:

(SEQ ID NO: 30) CACCCTCGCTGTTGGTAAACGTGCGCCTTGGGTATGTCCTCACCTGCATGATACGACATGTTGAAAAAGGTACAAGGCTGGGCGGATTTAAACAGTAGAATGAAAAGGTGCCACAAGAAAACTCGTCAAAGAATTGACTACGCGTCAATGTTCCATAGTTAAAAAGACTTGAACTCTGGATCAGGGACTTTCAAACAAGGATAGCTGCCTGGTCACCAGTCATTAACTGTAATGTAATGGCCATAGATGATGCATGAGTACAATAATAAAAAAACACCATCCAGCCAAATATATACTCCCTGTCACAAATGAAAATTCGTTTTAGATAATTAGTGGATTCATACAATATTTGTTGTATGTGTTTTATGTGTCTAGATTCATCATCCTCTATTTGAATATAGACAGAAAAATCATAACTAAAACGAATACTATTTGGGAACGGAGGGAGTACTACTTTGGCAGAATGCCCCCAGGAAAGTACCAGTTTCAGGGGTAGTTTGGAAGGCTAAACCTAGGGAGGGAAAACCCCCCACATGTAACTAAATATCTTATTCAAATGTTACCCCTAGGGATTACTCACCCTGGGAAATGAGAAGGGTCCCAAGGGGATTTCGGTTTCTATTATTTTTTCTGCAAACCATTTCAGAGCAATGATATGAAACCAAGCTAACTACTTATAACATTTCTTAAGAATATCAGACATAGGAAAGTGATGGCCTGGAACCAAAGTAAGACTGGTAGATAAATAGATCACTAGAATAAACCCTGACAGTTCATAGCCTTCATAGAAGCAAAAGGAAACACTACGGGAGCAATTGGTTGCTTGCACTAGCAATTCACTGCATTGGGTCTAATGCAGGATAGACTAAGCCAGCATAAGTGTGCGCAATGTGTTTGTGTTTGGTTGCCATGTTATAAGTAAGTTGCATTTGCTAATATctttctcctgactctaatgagtccacttttgctgactggtgggcgaaagtaagtaagcaagtgcacaaatccaaaagaagaggctttaacagtatcatcatcttgggggcttggtgtttatggcttcatcgtaataaggtggtttttgatggtgtcagtccttcaattattggcataaaggcaatttttttggatgaagttgaattctggaggcttgccggtgctaggcatcttgaggctttggttcctggtgctggaatttttaggtcaagggttcttttgggtgattagtgaagagcaggtgtgtgtggtctgctcgcactttttgttgttcgttctcctattgcgtgctgttgtttccaggcgcatttatggaggctgcagttttgtgcgcagcagaagttggtggttttgtgttttgtgttttgcctattttggcattgtactttggtccattttggactgttttcttctcttaatttaatgatgtgcagctctcctgcgcgtttaagaaaaaaaaaAGTTGGCTGTTTTGTATTTCTTGTGATCACCCATGCTTGTTGTGGTCAGATTAAACTCTCACGTTTAATGCTACAGAAGCATCCATGAGACAATGAAACACCGCTCAAAAGCCACGTAGTAGCATACCCTGACTTATGAATAAAGCAACTCGATCTGATTTATTTGAGAAAACAGGAAACTGACAAGTTATTTTTAACACAAAATTTCATTAAAAACGAATGGTAGACAATTACCAATCTGTAGGTCCCTGGCTTGCAAGTCCTCCCAATGTCTAAGAAATCAAATAGGAACTGCAGGCAAGCCAGCAAGAAAGTATTAATCACTGGATATAAAATATAAAGAAAAAAGAAGGAAAGACGGCTACTCGGCTAGCATATGTTTTTGTTAGGGGTGAAAATGGATACTTATTCAGAAATCATTTTTGATCTTTTTTCTTTAATTAGGAATAAATAGGATATAGAATATGCTAAGCAAATTCATATTCTTGTTCTTAGCATTGGGCTTGTAAAGATTCATAAAAGGTAAATCTCGAATTTATCATATATCTTAAATGGTAGATATAAAATTCAGATACAAATATTTTTCAACTTTTTTGTTGTAGGGAACAAATTATATTAAAAAAAATTATGCACAATTCTATTCTTATTTGTAATAATGTGCTTGATAACATAATAAAAGATTACCATCAAATTTCACACACACCCACCCACCCACCCACCCCTGCACGCACGCGCGCGCACACACACTATATGTGTGTtcaaacactgatagtttaaactgaaggcgggaaacgacaacctgatcatgagcggagaattaagggagtcacgttatgacccccgccgatgacgcgggacaagccgttttacgtttgg

The OB-4451 sequence (extended left border flank in 4588_652 is asfollows:

(SEQ ID NO: 31) gggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtTACCCACTTTCATCCCTAGTTTTTGTTCTGGATTCAAGCATCTCAAAATTGTTTACCTGAAGTTTATCAGTTTTGAGAAAGCGGCGCCCCTGTCGACTACCATCAGGCATTCGGACTACAACTGTCACAGCACCCTCTGCGTCTGGAGACGGTTCCGGTGGTAATGATGCTTGCTTCGAAGTGAGACTGGACTCTAGCTCCTATTTAATCAAAACATCAGGGACAACATGACAAATAGTAGTCAAATATCCAGGCAAGAAAAAAAAACCATAAACAATGAAAATACTGATCAAAAGTCCTGTTTGGATCTCCTAAGAAAAATGAGAATGAGATCCAAACAATTGGATTCTAGAATCCAGCTATCTATCCCAAACCCATTATTTGGCGAGATTTTCACTATGCAGAGGCAATGATCACTATAAGAATAAGATTCAAACACCCACTTATTATTTTTTTAATCCAGAAACCAGATTCTACATTCACTATAGAATCCAGAACTTCAATATGGGAATGAGATCCAAATAGACCCTAAGCCAAAATGAAATTGGTGAGATGAAGTGGCTAGTTGTCATAACCTCCTGTAAAGAAGACAGCGGTTTACAGTCCCAACACCCAAATAAACATGACATTAATATAATGACTACAACTCACAACCTAAACCTAAACCAATATACATCCAAACATAAGACAAAAGGAGAACTGAGTTTTATATGATCACACTGATGAACTGATGCTGTAGTCTAGCATTCAAGTGTTTAAGATAGTTGACTATAAACCCTTCACCTTGCAGATTACATGTGACAGAAAGATACCTCTTCCTCAAGTTGTTTTTTACGCCTTTCCTCCTCCTCTTGCTTCTGTTTCTCAAGAACAGCTTCTCTCGCAGCTGTTTCTTCAAGGCGACGGAGCTCAGCCTCCTGTAGGGCCTTTAACTCCTTTTCTTGATCAGCTTGTAGCGATGCAAGGTACTCATCGTCCTACAAATTTAAAATTTATAAAAGTGCTCACCCATAGTGGCAATTATGAACAATGGATAAATCTTAGACCTCAAACCTGCTGCTCTCGTAATAACCGCTGTTCAGTTAATGCTGGTGATGGAGAATGAGATATTGGGGGATAATAAGTAGAGGTTCTGTGAGAAGGCATAGAGAAAGGATATGTTGGTCCACCAAACATTGCAGCCTCAAGCATAACAGCTTCATCATGTTCCTCAGAAGAAATGCCACCCCACTTTATTGTAAAAAAAGACGTCAGAAATTAAACAAATCCATCTAATGTCTTAGCGCACATTGGAACCACAGATTATAATACCTCAGATGGGAAATCATCTCCATTATACTGGTGATTGTTCAGAACAGGGCTAGCACCTGGGTGCACTATTTTTGGTAATTCATTGTCCTCAGAAGGGGCACGCCGAGAACGACGCCTAACTAACGGCTGTTCTTCTACATCTTCAGCCTCCTCCTGGAAGCTCTCGTCATCTATTGGTTGCCTAGATGTTCCAGCCTTTCCTGAAGCTAGTCCTTGCCTCCAAAAGGAAATTGCATGTATAAGGAATCAAATGATACTGTAGTAGGGTAGCCTGAGTGAAGAGGTGGGTAGTAAAGTTAACATTCACCTCTCAACTATTCCATTTGCGGTTTCCATGCCTGCTTTATCAGAAGAATGGTCCCTCAAATTCACTTCCTCTTGATGCTGGCCTCCTTGCTCGACTATCTGATGATTATTGAAAAATTAGAGATGACATCAAGATAGGTTCAAGTAAGCATGTTGGGGA

Example 6. Feed Glucanase Expression in Subsequent Generations

Several “T1” progeny from original “T0” transgenic maize plants weregrown, and individual ears were either self-pollinated or pollinatedwith pollen from wild-type maize plants. Mature seed from the resultingears was then assayed for feed glucanase activity via the colorimetricassay. FIG. 12 illustrates that glucanase activity was observed in T1events. In this figure, the numbers along the abscissa correspond to theevent identifiers of the original T0 plants from which the progeny werederived. The highest activity was observed for seeds of T1 plant derivedfrom the 4597_69 event.

Example 7. Feed Glucanase Expression in Multi-Generations of Hemizygous,Homozygous and Hybrid Seeds

Progeny from original “T0” transgenic maize plants were grown andbackcrossed (pollinated with pollen from the wild type maize parents orpollinated onto the wild type parents) for 4 generations (in maizeinbred line E (BC4E), or in maize inbred line G (BC4G)). At eachgeneration, some individual ears were self-pollinated. PCR method wasapplied to select homozygous plants as described in Example 8. Hybridears were made by cross-pollinating transgenic line G plants withtransgenic line E plants, or vice versa.

FIG. 13 illustrates that glucanase activity in the hemizygous,homozygous and hybrid ears of event 4588.259. The homozygous and hybridears contained average activity of 190 units/g, which was approximatelydouble of ears from hemizygous plants.

Example 8. PCR Assays for Identifying and Determining Zygosity of theGlucanase Events 4588.259, 4588.652, and 4588.757

Maize glucanase events 4588.259, 4588.652, and 4588.757 carry transgenesthat result in seed-specific expression of glucanase enzyme. Event4588.259 originally carried two T-DNA insertions at independentlysegregating loci, but subsequently a single genetic locus was selectedfor propagation and development. Events 4588.652 and 4588.757 carry twoor more T-DNAs at a single genetic locus. Molecular identification andtracking of these transgenes can be done using standard PCR analysis(visually scoring an endpoint in a gel-based electrophoresis andstaining of the PCR products) or real-time PCR. In addition todetermining whether a plant is carrying a transgene, some of these PCRassays can also determine whether a plant is hemizygous (carrying onecopy of the insertion) or homozygous (carrying two copies of theinsertion).

FIG. 24 illustrates general real-time PCR assay design used to determineT-DNA locus presence (standard and real-time PCR) and zygosity(real-time PCR only).

In FIG. 14, the standard and real-time PCR assays include, for eachT-DNA locus, one primer (Primer A) that binds to a maize genomic regionthat is adjacent to where the T-DNA insert is located and one primer(Primer B) that binds to a region in the T-DNA that is close to PrimerA. To determine zygosity in the real-time PCR assay, a second referencegene (GWD, glucan water dikinase) is amplified (X and Y primers) alongwith the locus primers. Real-time PCR amplification of product fromPrimers A+B would indicate that the T-DNA locus is present and itsfluorescence relative to the GWD reference (ref) fluorescence fromamplification of product from Primers X+Y would determine whether it ishemizygous (one-copy) or homozygous (two-copy).

The standard multiplex PCR assay includes Primer A and B, as describedabove, but also another primer (Primer C) that binds to a maize genomicregion on the other side of the T-DNA, opposite Primer A, and would beclose to Primer A if the T-DNA insertion was not present, as in a wildtype (WT) locus. FIG. 15 illustrates general standard PCR assay designused to determine T-DNA locus presence and zygosity. When the T-DNAinsertion is present, the distance between Primer A and Primer C wouldbe too large to amplify a product under our PCR amplification conditionsand therefore absence of this amplification product is used to determinezygosity. PCR amplification of products from Primer A+B and Primer A+Cindicates that the T-DNA locus is present and is hemizygous (one-copy).PCR amplification of product from Primer A+B, but not Primer A+C,indicates that the T-DNA locus is present and is homozygous (two-copy).PCR amplification of product from Primer A+C only, indicates that noT-DNA is present and the plant is WT at this locus. Primers and probesfor all of these assays are listed in Tables 3 and 4.

TABLE 3 Standard and real-time (RT) PCR primers and probes usedto determine T-DNA locus presence andocus presence and zygosity of 4588.259, 4588.652, and 4588.757 eventsPrimer/ PCR Primer Probe Assay Event or Probe ID (type) Primer SequenceFluor* Quencher Standard/RT 4588.259 Primer 509 (A)GAATTGTTCATCATAAGGCGTGA (SEQ ID NO: 38) Standard/RT 4588.259 Primer516 (B) AACGTGACTCCCTTAATTCTCC (SEQ ID NO: 39) RT 4588.259 Probe PB5AAACTGAAGGCGGGAAACGACAAC HEX BHQ1 (SEQ ID NO: 40) Standard/RT 4588.652Primer 750 (A) GAGATGCTTGAATCCAGAACAAA (SEQ ID NO: 41) Standard/RT4588.652 Primer 751 (B) TTGTCTTGGTTGTGATGATGTG (SEQ ID NO: 42) Standard4588.652 Primer 749 (C) GATTACCATCAAATTTCACACACAC (SEQ ID NO: 43) RT4588.652 Probe PB17 TAGAACGACCGCCCAACCAGAC HEX BHQ1 (SEQ ID NO: 44)Standard 4588.757 Primer 513 (B) AAACGTCCGCAATGTGTTATT (SEQ ID NO: 45)Standard 4588.757 Primer 608 (C) TCATGCAATTGTGCCACC (SEQ ID NO: 46)Standard 4588.757 Primer 609 (A) ACATAGTCAACCTAACGGCTTAT (SEQ ID NO: 47)RT GWDref Primer 371 (X) GGTTATAAGCCCGGTTGAAGTA (SEQ ID NO: 48) RTGWDref Primer 525 (Y) CTATTCCTTGCTCGGACTGAC (SEQ ID NO: 49) RT GWDrefProbe PB2 CACCTGATATGCCAGATGTTCTGTCTCA FAM BHQ1 (SEQ ID NO: 50) *Fluor= fluorophore.

TABLE 4 4588.259, 4588.652, and 4588.757 event-specific PCR primercombinations and PCR product sizes Primer Primer Primer PCR ProductEvent A B C (bp) Assay Identifies 4588.259 509 516 137 T-DNA locus:OB-2880 (SEQ ID NO: 51) 4588.652 750 751 107 T-DNA locus: OB-4451 (SEQID NO: 52) 4588.652 750 749 174 WT locus (SEQ ID NO: 53) 4588.757 609513 100 T-DNA locus: OB-3237 (SEQ ID NO: 54) 4588.757 609 608 218 WTlocus: B73ref (SEQ ID NO: 55)

PCR assay using primers X (371) and Y(525) identifies the ZmGWDref locus(SEQ ID NO: 56).

DNA Extraction

These PCR assays will work with any DNA extraction method that yieldsDNA that can be amplified with PCR. A standard DNA extraction method(10X TE+Sarkosyl) that was used in this example is as follows: leaftissue (standard 1 cm hole punch) is sampled into a 96 deep-well block,metal beads are added, and the block is frozen at −80° C. for at least30 min. The block is then ground for 45 sec in a Kleco Pulverizer,centrifuged at 4,000 RPM for 3 min, the lid is removed, 300 μl of10XTE+Sarkosyl is added, the block is resealed, and the block is mixedat room temperature for 10-20 min. After incubation, the block iscentrifuged at 4,000 RPM for 5 min, 165 μl of upper aqueous phase isremoved and added to a 96-well PCR block, the PCR block is sealed, andthe block is incubated at 90° C. for 30 min. After incubation, 20 μl ofextract is added to 180 μl of sterile water in a 96-well plate (1:10dilution) to create the final DNA sample for PCR.

PCR

Events 4588.259, 4588.652, and 4588.757 standard and real-time PCRprimers are listed in Table 3 and standard PCR primer combinations withexpected PCR product sizes are listed in Table 4.

Standard PCR is performed with 2 μl of DNA extract and GoTaq (Promega)or Kapa 3G (Kapa Biosystems) PCR Mix in 30 μl reaction volumes with thefollowing components and conditions for each event:

Event 4588.259 Standard PCR:

Components (final concentration) were as follows: PCR Mix with buffer,MgCl₂, nucleotides, and enzyme (1×); primer 509 (400 nM) and primer 516(400 nM). Conditions were as follows: 95° C., 3 min; 33 cycles (95° C.,30 sec; 55° C., 30 sec; 72° C., 30 sec); 72° C., 8 min.

Event 4588.652 Standard PCR:

Components (final concentration) were as follows: PCR Mix with buffer,MgCl₂, nucleotides, and enzyme (1×), primer 749 (400 nM), primer 750(400 nM), and primer 751 (400 nM). Conditions were as follows: 95° C., 3min; 33 cycles (95° C., 30 sec; 55° C., 30 sec; 72° C., 30 sec); 72° C.,8 min.

Event 4588.757 Standard PCR:

Components (final concentration) were as follows: PCR Mix with buffer,MgCl₂, nucleotides, and enzyme (1×), primer 513 (400 nM), primer 608(400 nM), and primer 609 (400 nM). Conditions were as follows: 95° C., 3min; 33 cycles (95° C., 30 sec; 55° C., 30 sec; 72° C., 30 sec); 72° C.,8 min.

Standard PCR was analyzed by running approximately 15 μl of PCR producton a 3% agarose gel at 95V for 30 min. An example of results from astandard PCR analysis of the 4588.652 selfed segregating plants is shownin FIG. 16. Referring to this figure, ten PCR reactions from 10independent plants were separated on a 3% agarose gel stained withethidium bromide. Expected locus and zygosity band sizes are indicatedon the right side of the image.

Locus presence and zygosity is scored by visualizing specific bands ineach lane.

Real-Time PCR was performed with 2 μl of DNA extract in 20 μl reactionvolumes with the following components and conditions for each event:

Event 4588.259 Real-Time PCR:

Components (final concentration) were as follow: PCR Mix with buffer,MgCl₂, nucleotides, and enzyme (1×), primer 509 (400 nM, primer 516 (400nM), primer 371 (400 nM), primer 525 (400 nM), probe PB5 (200 nM) andprobe PB2 (200 nM). Conditions were as follows: 95° C., 4 min; 40 cycles(95° C., 5 sec; 60° C., 45 sec)

Event 4588. 652 Real-Time PCR:

Components (final concentration) were as follows: PCR Mix with buffer,MgCl₂, nucleotides, and enzyme (1×), primer 750 (400 nM), primer 751(400 nM), primer 371 (400 nM), primer 525 (400 nM), probe PB17 (200 nM)and probe PB2 (200 nM). Conditions were as follows: 95° C., 4 min; 40cycles (95° C., 5 sec; 60° C., 45 sec)

Event 4588. 259 Real-Time PCR. Real-Time PCR can be analyzed by anyreal-time PCR machine and software capable of four-channel fluorescencedetection. A Bio-Rad CFX96 real-time PCR machine and CFX ManagerSoftware were used to run an example of the 4588.259 real-time PCR assayon a selfed segregating population of 4588.259 plants. FIG. 17illustrates an example of real-time PCR data for 4588.259 to determinelocus presence and zygosity. In this figure, “RFU” refers to relativefluorescence units; “ntc” refers no target control. Presence of the4588.259 locus and zygosity was scored by the clustering of data pointson the graph.

Example 9. Germination Rates Among Seed from Independent TransgenicPlants that Express Feed Glucanase

Silks on wild-type plants were pollinated with pollen from individualtransgenic plants (WT×Transgenic), or silks on transgenic plants werepollinated with pollen from wild-type plants (Transgenic×WT). Mature,dried seed were collected from the resulting ears and planted into soil.Following 1-2 weeks of incubation, germination rates were calculated. Insome cases, this test was repeated following a second generation ofgrowth and pollination (T2). Examples of results from such germinationtests are shown in Table 5.

TABLE 5 Germination rates among seed expressing beta glucanase Germi- WT× Transgenic × nation Vector Event Transgenic WT Generation Sow % 458854 x T1 50 92 4588 17 x T1 50 82 4588 11 x T1 30 80 4588 161 x T1 15 674588 162 x T1 11 27 4588 215 x T1 13 62 4588 219 x T1 10 80 4597 18 x T130 37 4597 54 x T1 10 100 4597 56 x T1 10 100 4597 69 x T1 14 14 4597 69x T1 30 73 4588 161 x T1 20 100 4597 101 x T1 33 90 4597 104 x T1 30 704588 259 x T2 17 100 4588 251 x T2 17 100 4588 54 x T2 17 47 4597 101 xT2 20 100 4597 104 x T2 20 100

The germination rate in T1 and/or T2 for events 4597_54, 4597_56,4588_161, 4588_252, 4588_259, 4597_101, and/or 4597_104 was observed tobe 100%.

Example 10. Survival of Feed Glucanase Activity During Preparation ofPoultry Feed Pelleting

Milled grain from transgenic plants expressing the feed glucanase wasmixed with starter and grower corn-soy diets that were formulated forbroiler chickens. The basal corn-soy diet for starter broilers wascomposed as follows: 54.89% corn 08-2012, 32.81% soybean oilcake, 5.00%distillers dry grains plus soluble solids, 2.00% vermiculite, 1.99%dicalcium phosphate, 1.00% poultry fat, 0.81% limestone fine, 0.50%plain salt (NaCl), 0.20% DL-methionine, 0.20% choline chloride 60, 0.20%mineral premix, 0.13% L-lysine, 0.12% L-threonine, 0.05% vitamin premix,0.05% coban, and 0.05% selenium premix. The basal corn-soy diet forgrower broilers was composed as follows: 58.53% corn, 26.63% soybeanoilcake, 8.00% distillers dry grains plus soluble solids, 2.00%vermiculite, 1.69% dicalcium phosphate, 1.00% poultry fat, 0.76%limestone fine, 0.50% plain salt (NaCl), 0.20% mineral premix, 0.20%choline chloride 60, 0.13% DL-methionine, 0.13% L-lysine, 0.08%L-threonine, 0.05% vitamin premix, 0.05% coban, and 0.05% seleniumpremix.

Fine corn was ground using the hammermill screens: no. 4/4 for thestarter diet and no. 6/6 for the grower and finisher diets. Coarse corn(5% of total corn) was ground with the roller mill with 0/100 gapopenings.

Four diets were formulated for the pelleting trial as follows: Diet Awas a basal diet, Diet D was the basal diet mixed with a control enzyme,Diet E was the basal diet mixed with the milled transgenic corn graincontaining a high level of glucanase, and Diet F was the basal dietmixed with the milled transgenic corn grain containing a low level ofglucanase.

Milled grain from transgenic plants expressing the feed glucanase wasmixed with the basal diets at a ratio of approximately 1 lb transgenicgrain per 2000 lbs basal diet mixture. For the low dose diet, transgenicgrain was first mixed with non-transgenic grain at a weight ratio of 1:4(1 gram of transgenic grain per 4 grams non-transgenic grain) to dilutethe enzyme concentration prior to adding this ingredient to the basaldiets.

All feed diets were pelleted at 175-180° F. into 4.4 mm pellets, and thestarter diets were crumbled.

FIGS. 18A and 18B illustrate glucanase activity before and afterpelleting in the Grower Diet (FIG. 18A) and the Starter Diet (FIG. 18B).Referring to these figures, samples from the resulting mixture were thenremoved before and after pelleting of the feed. These samples were thentested via the colorimetric glucanase assay to determine whether theenzyme survived the pelleting process. The identity of the various dietsshown in FIGS. 18A and 18B were as follows: A, basal control diet (noexternal enzyme); D, positive control diet (commercially availableenzyme added); F, low-dose diet (including milled grain from plants thatexpress the feed glucanase); E, high-dose diet (including milled grainfrom plants that express the feed glucanase). It was observed thatglucanase activity was high in the high-dose diet for both the StarterDiet and the Grower Diet and survived pelleting.

Example 11. Thermal Stability of Grain-Expressed Feed Glucanase

Feed glucanase was prepared via microbial expression and purification,and suspended in SEC buffer (100 mM MES, 300 mM NaCl, pH6.3). Fivemicroliters of this preparation was mixed with 20 mg milled grain fromwild-type maize. In parallel, 5 μl of SEC buffer was mixed with 20 mgmilled grain from transgenic plants that express feed glucanase.Replicates from each of these two sets of samples were incubated at 94°C. or −130° C. for various periods of time, then allowed to cool to roomtemperature. Subsequently, residual glucanase activity was measured viathe colorimetric assay. FIGS. 19 A and 19B illustrate glucanase activityafter heat treatment. FIG. 19A illustrates glucanase activity aftertreatment at 130° C. FIG. 19B illustrates glucanase activity aftertreatment at 94° C. In these experiments, more activity survived whenthe feed glucanase was produced in the grain itself than when it wasadded to the grain exogenously. This finding demonstrates thatexpression and accumulation of the enzyme in grain effectively providesthe enzyme with additional thermal stability relative to the same enzymethat is produced microbially. In this particular instance both thegrain-expressed and the microbially-expressed enzymes have the sameprimary amino acid sequence. Therefore, the enhanced thermal stabilitythat was observed in the flour from transgenic grain is a function ofthe expression host.

Example 12. Activity of Microbially-Produced AGR2314 at Various pHValues

AGR2314 activity was measured at several pH values between 3 and 8.5.Each assay (500 μL) contained Britton-Robinson polybuffer (40 mM sodiumphosphate, 40 mM sodium borate, and 40 mM sodium acetate), 0.01% (v/v)Tween 20, one Beta-glucazyme substrate tablet (Megazyme, Wicklow,Ireland), and 20 nM of AGR2314 in a 2 mL Eppendorf tube. Samples wereincubated for 1 hour at 37° C. or 80° C. Reactions were terminated bythe addition of 1 mL of 2% (w/v) tris base. Samples were centrifuged at15,000×g for 10 minutes, and 100 μL of each supernatant (37° C. assays)or 5 μL supernatant plus 100 μL of water (80° C. assays) was transferredto a flat-bottomed 96-well microplate. Absorbances were read at 590 nm.Assays at each pH value were performed in triplicate. Single blankassays (containing no enzyme) were performed at each pH, and theseabsorbance values were subtracted from the assays containing enzyme.

FIGS. 20 and 21 illustrate the optimum pH for measuring AGR2314 activityat 37° C. (FIG. 20) and 80° C. (FIG. 21). Referring to FIG. 20, theoptimum pH was determined to be 7-7.5 at 37° C. FIG. 21 illustrates thatthe optimum was determined to be 6 at 80° C.

FIG. 22 shows an example of pH optimum of the feed glucanase that isproduced in transgenic flour.

To determine the relationship between the pH of the assay conditions andthe activity of the enzyme that was derived from transgenic grain, 5 mlof water containing 0.2% Tween-20 was mixed with 200 mg of flour fromtransgenic seed on a rotating platform for 1 hour at 60° C. Followingcentrifugation at 1500×g for 20 minutes in a clinical centrifuge, thesupernatant was transferred to a 15 mL Eppendorf tube. This sample wascentrifuged at 1500×g for 10 minutes in a tabletop centrifuge. Aliquotsof this protein extract were diluted 20-fold in assays to test each pHcondition by mixing 50 μl of extract with 950 μl of Britton-Robinsonpolybuffer (40 mM sodium phosphate, 40 mM sodium borate, and 40 mMsodium acetate) that had been prepared at pH 2-10. The pH of eachreaction mixture was checked using a pH strip. Five hundred microlitersfrom each mixture was transferred to a 96 deep-well plate for the assay.One beta-glucazyme tablet was added to each well and mixed by gentlevortexing, the plate was sealed and incubated at 80° C. for 1 hour. Thereactions were stopped by adding 1 mL of 2% (w/v) Tris-base to eachwell. The 96-well plate was centrifuged at 3000×g for 10 minutes in aclinical centrifuge, then 100 μL of the supernatant from each of thesamples was transferred to wells in a flat-bottom microplate, and theabsorbance at 590 nm was determined on a microplate spectrophotometer.As shown in FIG. 22, the seed-produced enzyme has a pH optimum betweenpH6 and pH7, but still retains a large fraction of its activity at a pHas high as 10.

Example 13. Activity of Microbially-Produced AGR2314 and AGR2414 onVarious Substrates

All reactions used 5 nM of AGR2314, AGR2414, or 5 μL of control enzymeat the indicated concentrations, in 200 mM sodium phosphate, 0.01% (v/v)Tween 20, pH 6.5. Reactions were carried out for one hour at either 37°C. or 80° C. and terminated as described.

Beta-glucosidase assays: the substrate was 1 mM pNP-D-glucopyranoside(Sigma Chemical Co. catalog # N:7006) and the positive control enzymewas Rhizobium etli beta-glucosidase (Prozomix, catalog No. PRO-E0110;315.9 Unit/mL). Reaction volumes were 500 μL; reactions were terminatedby the addition of 500 L of 2% (w/v) tris base. After centrifugation at3000×g for 10 minutes, 100 μL of supernatant was transferred to amicroplate and the absorbance at 405 nm was recorded.

Endocellulase assays: each assay contained one tablet of Cellazyme Csubstrate (Megazyme, catalog No, T-CCZ) in 500 μL buffer. Reactions wereterminated by the addition of 1 mL of 2% tris base. Samples werecentrifuged for 10 minutes at 15,000×g, 100 μL of supernatant wastransferred to a microplate, and the absorbance at 590 nm was recorded.

Exocellulase (cellobiohydrolase) assays: the substrate was 1 mMpNP-D-cellobioside (Sigma catalog No. N5759) and the positive controlenzyme was CBHI from Trichoderma longibrachiatum (Megazyme catalog No.E-CBHI; 0.5 Units/μL). Reaction conditions were as described above forthe beta-glucosidase assays.

Anylase assays: the substrate was Red Starch (Megazyme catalog No.S-RTAR; prepared as directed by the manufacturer) and the positivecontrol enzyme was α-amylase from Bacillus licheniformis (Megazymecatalog No. E-BLAAM: 3000 Units/mL). 245 μL buffer, 5 μL of enzyme, and125 μL of Red Starch reagent were mixed and incubated as describedabove. Reactions were terminated by the addition of 625 μL ethanol.After incubating at room temperature for 10 minutes, samples werecentrifuged for 10 minutes at 3000×g, and 100 μL of supernatant wastransferred to a microplate; absorbance at 510 nm was recorded.

Endoxylanase assays: each assay contained one tablet of Xylazyme AXsubstrate (legazyme catalog No, No. XAX-1000) and the positive controlenzyme was 100 mg/mL of Thermomyces lanuginosis xylanase (Sigma catalog#X2753) in assay buffer. Reactions were carried out as described abovefor the endocellulase assays.

Pectinase assays: the substrate was 25 mg/mL pectin (Sigma catalog No.P7536) in assay buffer and the positive control enzyme was pectinasefrom Aspergillus niger (Sigma catalog No. 17389) at 100 mg/mL in assaybuffer. Five p L of enzyme was added to 35 PL of pectin solution andincubated as described above. Reactions were terminated by the additionof 60 μL of DNS stop/reagent solution (Wicher et al. [2001], Appl.Microbiol. Biotechnol. 55, p. 578) followed by heating at 950° C. for 15minutes. Samples were centrifuged at 3000×g for 10 minutes, 20 μLsupernatant was mixed with 100 μL water in a microplate, and theabsorbance at 550 nm was recorded.

1,3-beta-glucosidase assays: each assay contained one tablet of1,3-beta-glucazyme HS substrate (Megazyme catalog No. ET-CUR200) and thepositive control enzyme was Trichoderma sp. 1,3-β-D-glucanase (Megazymecatalog No. E-LAMSE; 50 Units/mL). Reactions were carried out asdescribed above for the endocellulase assays.

1,4-beta-glucosidase assays: each assay contained one tablet ofBeta-glucazyme substrate (Megazyme catalog No. TBGZ-1000T). Reactionswere carried out as described above for the endocellulase assays, exceptthat 5 μL of supernatant was mixed with 100 PL of water in a microplatefor recording of absorbance.

FIGS. 23A and 23B illustrate the glucanase activity for hydrolyzingstarch, cellobiose (pNP-D-cellobioside), xylan (Xylazyme AX),HE-cellulose (Cellazyme C), barley-B-glucan (Beta-glucazyme), pectin andPNP-D-gluopyranoside at 37° C. (FIG. 23A) and 80° C. (FIG. 23B).Referring to these figures, it was observed that both AGR2314 andAGR2414 enzymes were highly active in hydrolyzing cellobiose andHE-cellulose at 37° C. and 80° C.

Example 14. Glucanase Activity on Seed Fiber

Glucose release from untreated seed fiber 20 mg seed fiber was digestedat pH 5.0 with 5 μM AGR2314 protein for 72 hours at 55 C. A commercialenzyme cocktail was used at full loading (FCT) as a positive control.After enzymatic hydrolysis, the soluble sugars in reaction supernatantwere hydrolyzed into monomers via acid hydrolysis at 121° C. FIGS. 24Aand 24B illustrate release of monomeric sugars after enzymatichydrolysis of the seed fiber. FIG. 24A shows glucose yield and FIG. 24Bshows xylose yield. Pre-acid hydrolysis (light gray bars) and total(dark gray bars) sugars were separated and quantified via HPLC using aBio-Rad Aminex HPX-87-P ion-exclusion column. AGR2314 does not releasemonomeric glucose or xylose from untreated seed fiber. However, thisenzyme was able to solubilize untreated seed fiber into oligosaccharideswhich account for approximately 80% of the total sugars released by acommercially-available cellulase-enzyme cocktail.

Glucose release from dilute acid-pretreated seed fiber 20 mg seed fiberwas pretreated at 80 C in 0.5% H₂SO₄ for 16 hours, then neutralized topH 5.0. The pretreated seed fiber was digested at pH 5.0 with 2 μMAGR2314 and a suite of glucanase, cellobiohydrolase, endoglucanase, andbeta-glucosidase (all at 2 μM loading) for 72 hours at 55° C. Acommercial enzyme cocktail (Accellerase XY, Genencor) was used at fullloading (FCT) as a positive control. After enzymatic hydrolysis, thesoluble sugars in reaction supernatant were hydrolyzed into monomers viaacid hydrolysis at 121° C. FIGS. 25A and 25B illustrate release ofmonomeric sugars after enzymatic hydrolysis of the seed fiber. FIG. 25Ashows glucose yield and FIG. 25B shows xylose yield. Pre-acid hydrolysis(light gray bars) and total (dark gray bars) sugars were separated andquantified via HPLC using a Bio-Rad Aminex HPX-87-P ion-exclusioncolumn.

AGR2314 did not release sugars from pretreated seed fiber at greaterlevels than the pretreatment itself. When combined with other cell-walldegrading enzymes, approximately 90% of total sugars were released ascompared to a commercially-available cellulase-enzyme cocktail.

Example 15. The Use of Glucanase Enzymes on Broiler Live Performance

The chemical energy contained within an animal's diet, and itsavailability to the animal eating the diet, are critical characteristicsinfluencing the nutritional value of any diet. Diets rich in energy,provide adequate nutrition and promote rapid growth to higher levelsthan diets that are deficient in energy. Therefore, determining theenergy within a diet, and altering energy availability by usingglucanase enzymes, provides an important set of tools to improve animalnutrition and therefore animal performance.

To demonstrate the use of glucanase enzymes in broiler production,metabolizable energy and nutrient digestibility, male broilers were fedwith alternative feed ingredients (wheat, barley and low-fat DDGS) withor without supplemental glucanase. Broiler body weight gain, feedconsumption and feed conversion rate were determined and feed glucanaseenzyme was evaluated.

Dietary Treatments and Procedures

Day-old male broiler were obtained from a commercial hatchery andrandomly allocated to 64 battery cages in groups of 10. Experimentaldiets were fed from 0 to 28 d of ages. Initial group weights wereobtained and equalized amongst the treatments. Feed disappearance andbody weight were measured weekly (7, 14, 21, and 28 d of age) tocalculate live performance parameters (feed consumption, body weightgain, and feed conversion ratio). In addition, excreta were collectedtwice to determine apparent metabolizable energy (AME) of the diets at14 and 29 d of age.

Dietary treatments were fed in a 4×2 factorial design and furtherdelineated below. Four different diets (corn/soybean meal based,corn/wheat based, corn/barley based, and corn/LF-DDGS based diets) andtwo levels of glucanase (with or without) were fed. Diets wereformulated to be isocaloric and all nutrients, with the exception ofenergy, were formulated to meet or exceed the nutrient requirements. Theenzyme treatments had the enzyme added on top of the diet. In addition,titanium dioxide was added to the diets as an indigestible marker todetermine AME and nutrient digestibility. Table 6 describes dietarytreatments.

TABLE 6 Treatment delineation Trt Diet Enzyme 1 Corn-soybean meal − 2Corn-soybean meal + 3 Corn-soybean meal-wheat − 4 Corn-soybeanmeal-wheat + 5 Corn-soybean meal-LF- − DDGS 6 Corn-soybean meal-LF- +DDGS 7 Corn-soybean meal-barley − 8 Corn-soybean meal-barley + Total 640male broilers Total Males needed: 8 treatments × 8 reps × 10 birds/cage= 640 male broilers + 60 for equating = 700 male broilers

Temperature

Battery cages: 92° F. from placement to 4 d, 90° F. from 5 to 9 d, 84°F. from 10 to 15 d, 80° F. from 16 to 24 d, 78° F. from 25 to 29 d,

Room Setup

Prior to bird placement, lighting and temperature in the battery cagerooms were set 48 hours in advance. Wire bottoms were added to thebattery cages until day 0 to 4. Unless noted in the schedule, forcollection periods, excreta pans were scrapped on a regular basis toavoid any pest and odor issues.

Lighting Program

A 23 light:1 dark with a lighting intensity of 3.0 ft² was implementedfrom placement until 7 d of age. A 23 light: 1 dark lighting schedulewas implemented from 8-21d with a lighting intensity of 1.0 ft candles.A 23 light: 1 dark lighting schedule was implemented from 22-28 d of agewith a lighting intensity of 0.3 ft candles.

Special Instructions

Wire bottoms were placed into all battery cages prior to the start oftheexperiment or day-old chicks will fall through the floors. Wire bottomswere removed at d 7. In addition, cage doors were modified toaccommodate smaller birds from 0 to 7 d of age.

Any mortality was not replaced.

All diets were fed in mash form. Mix sheets were forthcoming.

To avoid cross contamination of the enzyme, separate feed scoops wereneeded. Table 7 describes experimental timeline.

TABLE 7 Timeline of dietary treatments Age of Day birds ExperimentalTimeline Lighting Temperature Tue −2 23L: 1D 92° F. 3.0 ft² Thu 0 Place720 male broilers in battery cages Equate weights Start experimentaldiets Tue 5 90° Thu 7 Weigh all birds and feed Remove wire bottoms Fri 823L: 1D 1.0 ft² Sat 9 Sun 10 84° Mon 11 Clean excreta pans Start 14 dcollection period Thu 14 Weigh all birds and feed Collect excreta for 14d collection period Sat 16 80° Sun 17 Wed 20 80°^(B-east) Thu 21 Weighall birds and feed Fri 22 23L: 1D 0.3 ft² Tues 25 Clean excreta pans 78°Start d 29 collection period Fri 28 Weigh all birds and feed 75° Collectexcreta for 29 d collection period END OF EXPERIMENT

FIG. 26 illustrates the body weight gain (BWG) during the 28-day poultryfeeding trial. Referring to FIG. 26, four different diets, corn/soybeanmeal based, corn/barley based, corn/wheat based, and corn/LF-DDGS based,with (+) or without (−) a glucanase were tested. Still referring to FIG.26, average of BWG with or without glucanase across the four diets wasshown. It was observed, that body wait gain was on average higher inchickens fed with the diets that included a glucanase.

FIG. 27 illustrates the changes in poultry BWG per time interval during28 day feeding trial. Referring to FIG. 27, initial group weights wereobtained and equalized amongst the treatments. BWG was measured weekly(7, 14, 21, and 28 d of age) for broilers feed using the corn/LF-DDGSdiet with (+) or without (−) a glucanase. Still referring to FIG. 27, itwas observed across all treatments that body wait gain in chickens fedwith the glucanase including diets was higher than in chickens fed withdiets without glucanase.

FIG. 28 illustrates feed consumption during the 28-day poultry feedingtrial using two different diets (corn/barley based and corn/LF-DDGSbased) with (+) or without (−) a glucanase. Referring to FIG. 28, it wasfound that feed consumption was higher for the diets that included aglucanase.

FIG. 29 illustrates the feed conversion rate (FCR) during the 28-daypoultry feeding trial with two different diets (corn/barley based andcorn/LF-DDGS based diets) with (+) or without (−) a glucanase. The feedconversion rate refers to the feed consumption required for gaining thebody weight for a tested animal. The FCR is calculated by dividing thevalue of feed consumption by the value of body weight gain. Referring toFIG. 29, it was observed that the FCR was lower for the diets thatincluded a glucanase. These data indicates that diets that included aglucanase facilitated digestion and feed consumption in the testedanimals.

Example 16. The Use of Glucanase Enzymes for Broiler Live Performance

To demonstrate the effect of glucanase enzymes in broiler production,936 male broilers were feed with varies glucanase concentrations in a 17day battery trial. Birds were weighed at day 17. Feed compositionincludes corn, soybean meal and fat (soybean oil). Table 8 describesexperimental details of the 17 day battery trial.

TABLE 8 Experimental Treatments: (diets were fed through day 17)—totalof 9 TRTs Trt Code Description Dose 1 Positive Control (PC) — 2 NegativeControl — (NC, less 50-60 kcal/lb of PC) 3 NC + Industry Std Enzyme_1*0.25 lb/ton 4 NC + Industry Std Enzyme_2**  0.2 lb/ton 5 NC + Glucanase5 6 NC + Glucanase 50 7 NC + Glucanase 100 8 NC + Glucanase 250 9 NC +Glucanase 500 *Industry Std Enzyme_l refers to ENSPIRAT ™ (JBS United)**Industry Std Enzyme_2 refers to HOSTAZYM X ® (Huvepharma) No. oftreatments 9 Broilers per replicate 8 Replicates per treatment 13Broilers per treatment 104 Total No. of replicates 117 Total No. ofbroilers 936

Referring to FIG. 30, the inclusion of beta-glucanase in treatments 7(Trt_7) and 8 (Trt_8) significantly (p-value <0.05) increased bodyweight gain compared to the positive control (PC), negative control(NC), and treatments 3 (Trt_3) and 4 (Trt_4), which contained acommercial NSPase inclusion. The glucanase inclusion in treatments 5(Trt_5), 6 (Trt_6), and 9 (Trt_9) produced intermediate results.

REFERENCES

-   Leeson, S. and L. Caston. 2000. Commercial enzyme and their    influence on broilers fed wheat or barley. J. Appl. Poult. Res.    9:242-251.-   Yu, B. and T. K. Chung. 2004. Effects of multiple-enzyme mixtures on    growth performance of broilers fed corn-soybean meal diets. J. Appl.    Poult. Res.    13:178-182.

The references cited throughout this application, are incorporated forall purposes apparent herein and in the references themselves as if eachreference was fully set forth. For the sake of presentation, specificones of these references are cited at particular locations herein. Acitation of a reference at a particular location indicates a manner(s)in which the teachings of the reference are incorporated. However, acitation of a reference at a particular location does not limit themanner in which all of the teachings of the cited reference areincorporated for all purposes.

It is understood, therefore, that this invention is not limited to theparticular embodiments disclosed, but is intended to cover allmodifications which are within the spirit and scope of the invention asdefined by the appended claims; the above description; and/or shown inthe attached drawings.

What is claimed is:
 1. A transgenic plant, part or seed thereofcomprising at least one synthetic nucleic acid encoding a glucanase andat least one synthetic polynucleotide producing a diagnostic ampliconfor identifying event 4588.652, wherein the at least one nucleic acidcomprises a sequence with at least 90% identity to the sequence setforth in SEQ ID NO: 8, and the at least one synthetic polynucleotidecomprises a sequence with at least 90% identity to the sequence selectedfrom the group consisting of SEQ ID NOS: 29-31.
 2. The transgenic plantor part thereof of claim 1, wherein the glucanase is capable ofdegrading one or more polysaccharides selected from the group consistingof beta-glucan, cellulose, cellobiose, pNP-D-glucopyranoside and xylan.3. The transgenic plant or part thereof of claim 1, wherein theglucanase is active upon expression in the plant and exposure to a pH inthe range from 5.0 to 10.0.
 4. The transgenic plant or part thereof ofclaim 1, wherein the glucanase is active upon expression in the plantand exposure to a temperature in the range from 25° C. to 130° C.
 5. Thetransgenic plant or part thereof of claim 1, wherein the glucanaseactivity has improved stability upon expression in the plant compared tothe activity of a glucanase having an identical amino acid sequence andexpressed in a bacterial cell.
 6. The transgenic plant or part thereofof claim 1, wherein a plant is selected from the group consisting of:wheat, maize, barley, and sorghum.
 7. An animal feedstock comprising thetransgenic plant, part or seed thereof of claim
 1. 8. The animalfeedstock of claim 7 further comprising a feed supplement.
 9. The animalfeedstock of claim 8, wherein the feed supplement is plant materialselected from the group consisting of: a non-transgenic plant, anothertransgenic, mutant and engineered plant.
 10. The animal feedstock ofclaim 8, wherein the feed supplement comprises one or more exogenousenzymes selected from the group consisting of: a hydrolytic enzymeselected from the group consisting of: xylanase, endoglucanase,cellulase, protease, phytase, amylase and mannanase.
 11. The animalfeedstock of claim 8, wherein the feed supplement comprises at least onecomponent selected from the group consisting of: corn meal, cornpellets, wheat meal, wheat pellets, wheat grain, barley grain, barleypellets, soybean meal, soybean oilcake, sorghum grain and sorghumpellets.
 12. The animal feedstock of claim 8, wherein the feedsupplement comprises at least one component selected from the groupconsisting of: soluble solids, fat and vermiculite, limestone, plainsalt, DL-methionine, L-lysine, L-threonine, COBAN®, vitamin premix,dicalcium phosphate, selenium premix, choline chloride, sodium chloride,and mineral premix.
 13. A method of producing an animal feedstockcomprising mixing the transgenic plant, part or seed thereof of claim 1with other plant material to form a mixture.
 14. The method of claim 13,wherein the method further comprises pelletizing the mixture.
 15. Amethod of increasing metabolizable energy of a diet, wherein the methodcomprises mixing the transgenic plant, part, or seed thereof of claim 1with a feed ingredient.
 16. The method of 15, wherein the feedingredient comprises at least one component selected from the groupconsisting of: corn meal, corn pellets, wheat meal, wheat pellets, wheatgrain, wheat middlings, barley grain, barley pellets, soybean meal, soyhulls, dried distillers grain, soybean oilcake, sorghum grain andsorghum pellets.
 17. The method of claim 15, wherein the feed ingredientcomprises at least one component selected from the group consisting of:soluble solids, fat and vermiculite, limestone, plain salt,DL-methionine, L-lysine, L-threonine, COBAN®, vitamin premix, dicalciumphosphate, selenium premix, choline chloride, sodium chloride, mineralpremix, and one or more exogenous enzymes.